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Novel YAP1 Activator, Identified by Transcription-based Functional Screen, 1

Limits Multiple Myeloma Growth 2

3

Junichi Maruyama1,#, Kazutoshi Inami1,#, Fumiyoshi Michishita1,#, Xinliang Jiang1, 4

Hiroaki Iwasa1, Kentaro Nakagawa1, Mari Ishigami-Yuasa2, Hiroyuki 5

Kagechika2,3, Norio Miyamura4, Jun Hirayama4, Hiroshi Nishina4, Daichi 6

Nogawa5, Kouhei Yamamoto5, Yutaka Hata1,6 7

8

1Department of Medical Biochemistry, Graduate School of Medical and Dental 9

Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 10

2Chemical Biology Screening Center, 3Institute of Biomaterials and 11

Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan 12

4Department of Developmental and Regenerative Biology, Medical Research 13

Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan. 14

5Department of Comprehensive Pathology, Graduate School of Medical and 15

Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 16

6Center for Brain Integration Research, Tokyo Medical and Dental University, 17

Tokyo 113-8519, Japan. #These authors equally contributed to this work. 18

19

Running title: YAP1 Activator Suppresses Multiple Myeloma Cell Viability. 20

Abbreviations: ABL1, Abelson murine leukemia viral oncogene homolog 1; 21

APMSF, (Amidinophenyl)methanesulfonyl fluoride; EMT, epithelial-mesenchymal 22

transition; GFP, green fluorescent protein; LATS, large tumor suppressor; LDH, 23

Lactate dehydrogenase; MM, multiple myeloma; NIQR, normalized interquartile 24

range; PP, protein phosphatase; qRT-PCR, quantitative reverse 25

transcription-PCR; TEAD, TEA-domain family proteins; YAP1, Yes-associated 26

protein 1. 27

Financial support: H. Iwasa (JSPS, 26460359); Y. Hata (JSPS, 26293061; the 28

Mitsubishi Foundation, 26138) 29

Correspondence author: Yutaka Hata, Department of Medical Biochemistry, 30

Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental 31

University, Tokyo 113-8519, Japan. E-mail:[email protected] 32

Research. on December 22, 2020. © 2017 American Association for Cancermcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 23, 2017; DOI: 10.1158/1541-7786.MCR-17-0382

http://mcr.aacrjournals.org/

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1

Abstract 2

Yes-associated protein 1 (YAP1) interacts with numerous transcription factors 3

including TEA-domain family proteins (TEAD) and p73. YAP1 is negatively 4

regulated by the tumor suppressor Hippo pathway. In human cancers, the 5

deregulation of the Hippo pathway and YAP1 gene amplification lead to the 6

activation of YAP1, which induces epithelial-mesenchymal transition (EMT) and 7

drug resistance. YAP1 inhibitors are expected to be useful in cancer therapy. On 8

the other hand, in certain cancers, YAP1 up-regulates p73-dependent gene 9

transcription and behaves as a tumor suppressor. Moreover, as YAP1 regulates 10

self-renewal and differentiation of tissue stem cells and plays an important role in 11

tissue homeostasis, YAP1 activators may contribute to the regenerative 12

medicine. With this in our mind, we screened for YAP1 activators by using 13

human retinal pigment epithelial ARPE-19 cells expressing the 14

TEAD-responsive fluorescence reporter under the co-expression of YAP1. From 15

an extensive chemical compound library (n=18,606) 47 candidate YAP1 16

activators were identified. These compounds were characterized to determine 17

whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 18

activator was effective against the human multiple myeloma IM-9 cells and 19

chronic myeloid leukemia K562 cells. 20

21

Implications: YAP1 activation limits growth, induces apoptosis, and may be 22

useful at suppressing hematological cancers. 23

24

*Recent article that might be of interest to the manuscript* 25

Ehmer U and Sage J, Control of Proliferation and Cancer Growth by the Hippo 26

Signaling Pathway. Mol Cancer Res, 2016 14(2)127-40. DOI: 27

10.1158/1541-7786.MCR-15-0305.. Recent review discusses Hippo/Yap 28

signaling in growth and proliferation in cancer. 29

30

Keywords: ABL1, the Hippo pathway, tumor suppressor, YAP1. 31

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Introduction 1

Yes-associated protein 1 (YAP1) was identified as a protein interacting with yes 2

tyrosine kinase(1). Thereafter, many YAP1-interacting proteins, such as ErbB4, 3

p73, TEAD, SMAD, and Runx2, were reported(2-6). YAP1 does not directly 4

bind to DNA but regulates gene transcription through the interaction with these 5

molecules. YAP1 is negatively regulated by the tumor suppressor Hippo 6

pathway(7,8). YAP1 is phosphorylated by large tumor suppressor kinase 7

(LATS) 1 and 2, the core kinases of the Hippo pathway. The phosphorylated 8

YAP1 is recruited from the nucleus to the cytoplasm, and undergoes degradation. 9

In human cancers, the Hippo pathway is frequently deregulated and YAP1 gene 10

is amplified, so that YAP1 activity is enhanced(9). YAP1 up-regulates cell 11

cycle-promoting and anti-apoptotic genes(10). Cancer cells with hyperactive 12

YAP1 undergo epithelial-mesenchymal transition and acquire drug 13

resistance(10). The activity of YAP1 correlates with short survival in cancer 14

patients. Hence, YAP1 is regarded as a therapeutic target in cancer 15

therapy(11). Among transcription factors interacting with YAP1, TEAD is the 16

most important in the induction of epithelial-mesenchymal transition and drug 17

resistance(12,13). Accordingly, verteporfin and synthetic peptides that block 18

the interaction between YAP1 and TEAD are shown to suppress cancer 19

growth(14-16). 20

The role of YAP1 in cancer is twisting. YAP1 co-operates with p73, 21

up-regulates pro-apoptotic genes, and suppresses certain cancers(17). How 22

YAP1 determines which fate as an oncoprotein or as a tumor suppressor to 23

adopt is not fully understood, but the accumulating evidence suggests that 24

Abelson murine leukemia viral oncogene homolog 1 (ABL1) is a key 25

determinant(18). Upon DNA damage, ABL1 phosphorylates YAP1 at tyrosine 26

357 and promotes the interaction with p73. A recent study has revealed that 27

multiple myeloma (MM) cells show DNA damage response but escape cell death, 28

because YAP1 activity is low(19). When YAP1 is exogenously expressed or the 29

Hippo pathway is suppressed to enhance YAP1 expression, MM cells die via the 30

ABL1-YAP1-p73 axis. We can surmise that in cancers with the high expression 31

of ABL1 and with the low expression of YAP1, YAP1 activators trigger apoptosis 32




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and are therapeutically effective. 1

YAP1 plays a crucial role in the regulation of tissue stem cells(20). 2

YAP1 is essential for tissue repair and protection in intestine, liver, heart, and 3

skin(21-26). YAP1 is also necessary to maintain neural stem cells in brain(27). 4

Based on these properties of YAP1, we infer that YAP1 activators are useful in 5

the regenerative medicine. 6

We previously searched for YAP1 modulators by means of human 7

osteosarcoma U2OS cells expressing green fluorescent protein (GFP)-tagged 8

YAP1(28). We used the subcellular localization of GFP-YAP1 as the read-out 9

and found that dobutamine inhibits YAP1 through -adrenergic receptor. 10

However, we have not yet found the compounds that activate YAP1. In this 11

study, we established a new cell-based assay, in which the YAP1-dependent 12

TEAD-responsive reporter activity is monitored. We performed a small 13

chemical compound library screening with the use of this assay and obtained 14

candidate YAP1 activators. We characterized these compounds to examine 15

whether they indeed activate YAP1 and tested the idea that YAP1 activator can 16

be used against MM and other blood cancer cells. 17

18

Materials and Methods 19

20

DNA constructions and virus productions. 21

22

pCIneoFLAG-His6 (pCIneoFH), pClneoFLAG-His6-FLAG (pClneoFHF), 23

pCIneoMyc, pCIneoLuc, pCIneomCherry, and pQCXIP-EF were described 24

previously(28-32). NheI/EcoRI fragment was isolated from pEGFP-C2 25

(Clontech) and ligated into NheI/EcoRI sites of pCIneo (Promega) to generate 26

pCIneoEGFPC2. pIRES2-EGFP (Clontech) was cut with NotI, filled in, and 27

digested with NheI. pLL3.7 vector was cut with EcoRI, filled in, and digested 28

with NheI. The fragment from pIRES-EGFP was ligated into pLL3.7 to replace 29

GFP with IRES-GFP and the resulting vector was named pLL3.7 K122. PCR 30

was performed with the primers (H3244, 5’-caattggcagaaatcggtactggctttccatc-3’ 31

and H3245, 5’-acgcgtgaattccgcgttatcgctctgaaagta-3’) on pHTN-Halo-tag® vector 32




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(Promega) to amplify Halo-tag. The PCR product was digested with MfeI/MluI 1

and ligated into EcoRI/MluI of pCIneomCherry to generate pCIneomCherry-Halo. 2

pCMV SPORT human YAP (MHS1010-7508628) was purchased from Open 3

Biosystems. The coding region was amplified by PCR with the primers (H2130, 4

5’-acgcgtcccgggcagcagccgccgcctcaa-3’ and H2352, 5

5’-gatatcataaccatgtaagaaagctt-3’), digested by MluI/EcoRV and ligated into 6

MluI/Smal sites of pCIneoFH to generate pCIneoFH-YAP1. MluI/NotI fragment 7

from pCIneoFH-YAP1 was ligated into MluI/NotI sites of pCIneoLuc and 8

pCIneomCherry-Halo to generate pCIneoLuc-YAP1 and 9

pCIneomCherry-Halo-YAP1. NheI/NotI fragment from 10

pCIneomCherry-Halo-YAP1 was ligated into XbaI/NotI of pQCXIP-EF to 11

generate pQCXIP-mCherry-Halo-YAP1. pCIneoFH-YAP1 was digested with 12

NotI, filled in, and cut with NheI. The isolated fragment was ligated into 13

NheI/SmaI sites of pLL3.7 K122 to generate pLL3.7 K122 FH-YAP1. NcoI/BglII 14

from pEYFP-C1 (Clontech) was ligated into NcoI/BamHI of pGL3 to replace 15

luciferase with YFP and to generate YFP-reporter. BamHI/Sall and Sall/HindIII 16

fragments from 8xGT-IIC-δ51LucII luciferase reporter (a gift from Hiroshi Sasaki) 17

(RIKEN RDB08067)(33) were ligated Into BglII/HindIII sites of the YFP-reporter 18

to generate pTEAD-responsive-promoter YFP, which was digested with NotI and 19

partially digested with HindIII. The resulting fragment and HindIII/XbaI 20

fragment from H2B-mCherry (a gift from Robert Benezra) (Addgene plasmid, 21

20972)(34) were ligated into NotI/XbaI sites of pLL3.7 K122 FH-YAP1 and 22

pLL3.7 K122 to obtain pLL3.7 K122 23

FH-YAP1-ires-GFP-TEAD-responsive-promoter-H2B-mCherry reporter (Fig. 1A) 24

and pLL3.7 K122 control-TEAD-responsive-promoter-H2B-mCherry, respectively. 25

pCMV FLAG-human YAP1 was described previously(35). pCIneoMyc- and 26

pCIneoFLAG-LATS1 were generated from pCGN HA-Warts (a gift from Hiroyuki 27

Saya) using PCR with the primers (H1921, 5’-acgcgtatgaggcctaagacctttcc-3’ and 28

H1922, 5’-gtcgactaaacatatactagatcgcga-3’), while pCIneoMyc-LATS2 was 29

generated from pcDNA LATS2-FLAG (a gift from Tadashi Yamamoto) with the 30

primers (H1923, 5’-acgcgtatgaggccaaagacttttcc-3’ and H1924, 31

5’-gtcgactacacgtacacaggctggc-3’). pCIneoLuc-PP1A and pCIneoLuc-PP2A 32




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were described previously(30). pCMV SPORT human TEAD4 1

(MHS1010-58163) was purchased from Open Biosystems. PCR was 2

performed with the primers (H2710, 5’-gaattcgagggcacggccggcaccat-3’ and 3

H2710, 5’-gtcgactcattctttcaccagcctg-3’). The PCR product was ligated into 4

EcoRI/Sall sites of pCIneoLuc to generate pCIneoLuc-TEAD4. pCMV alkaline 5

phosphatase is a gift from Sumiko Watanabe. PCR was performed with the 6

primers (H3159, 5’-gcggccgcttaagtgaacaactagtgcca-3’ and H3160, 7

5’-caattgagatctttcacaaattttgtaatc-3’) on piLenti-siRNA-GFP (Applied Biological 8

Materials Inc.) to amplify GFP-2A-puromycin. The product was digested with 9

SpeI/MfeI and ligated into NheI/EcoRI of pLL3.7 to generate 10

pLL3.7-GFP-2A-puro. PCR was performed with the primers (H3161, 11

5’-gcggccgcccccttcaccgagggcctattt-3’ and H3162, 12

agatctagactattctttcccctgcactgt-3’ ) on pLKO1-shYAP2 (a gift from Kunliang 13

Guan) (Addgene plasmid, 27369)(6). The product was digested with NotI/XbaI 14

and ligated into the same sites of pLL3.7-GFP-2A-shYAP2. Human p73 cDNA 15

was obtained from Open Biosystem (40125802). The coding region was 16

amplified by PCR with the primers (H2074, 5’-acgcgtatggcccagtccaccgccac-3’ 17

and H2075, 5’-gtcgactcagtggatctcggcctcc-3’), digested by MluI/Sall and ligated 18

into pCIneoFHF to generate pCIneoFHF-p73. pGL2 MDM2 reporter was a gift 19

of Hitoshi Okazawa (Tokyo Medical and Dental University)(36). PCR was 20

performed with the primers (H2781, 5’-gacgtcgacgtgcggtctctctctgtt-3’ and H2782, 21

5’-gcggccgctattctagaaattcagggccgggattctc-3’) on piPSC-Nanog (SBI System 22

Biosciences) to amplify 2A self-cleaving peptide and with the primers (H2862, 23

5’-gctagccccaacatgcctgaaccctctaagtct-3’ and H2863, 24

5’-gtcgaccttgtacagctcgtccatgccgcc-3) on H2B-mCherry. The former product 25

was digested with SalI/NotI and ligated into the same sites of pCIneoFH to 26

generate pCIneoFH-2A, and the later product was digested with NheI/Sall and 27

ligated into the same sites of pCIneoFH-2A to generate 28

pCIneo-H2B-mCherry-2A. SmaI/NotI fragment from pDsRed-N1 (Clontech) 29

was ligated into the same sites of pIRES (Clontech) to generate 30

pIRES-DsRedN1. NheI/PmlI fragment from pLL3.7 K122 and PmlI/MfeI 31

fragment from pIRES-DsRedN1 were ligated into NheI/EcoRI sites of pLL3.7 to 32




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generate pLL3.7-ires-DsRedN1. NheI/NotI fragment from 1

pCIneo-H2B-mCherry-2A was ligated into the same sites of 2

pLL3.7-ires-DsRedN1 to generate pLL3.7-H2B-mCherry-2A. PCR was 3

performed with the primers (H3112, 5’-tctagactagttggtaaagccaccatggaa-3’ and 4

H3113, 5’-ctagtcgacggcgatctttccgcccttctt-3’) on pGL3 (Promega) to amplify 5

luciferase. The product was digested with XbaI/NotI and ligated into the same 6

sites of pLL3.7-H2B-mCherry-2A to generate 7

pLL3.7-H2B-mCherry-2A-luciferase. 8

Antibodies and reagents. Rat monoclonal anti-YAP1 was described 9

previously(28). Other antibodies and reagents were obtained from commercial 10

sources: mouse anti-PARP (51-6639GR), and mouse anti-BAX (610982) (BD 11

Pharmingen); mouse anti--tubulin (T9026), mouse anti--actin (A1978), 12

Hoechst 33342, propidium iodide, trypan blue solution, thiazolyl blue tetrazolium 13

bromide, and SIGMAFASTTM 3,3’-diaminbenzidine tablets (D4293) 14

(Sigma-Aldrich); anti-DYKDDDDK-tag antibody (014-22383), 15

anti-DYKDDDDK-tag beads (016-22784), Phos-tagTM acrylamide and imatinib 16

(Wako Pure Chemical Industries); mouse anti-phospho-Histone H2A.X (Ser139) 17

(05-636) (Merck Millipore); rabbit monoclonal anti-YAP1 (14074), rabbit 18

monoclonal anti-YAP1/TAZ (D24E4) (8418), rabbit polyclonal anti-phospho-S127 19

YAP1 (4911), rabbit polyclonal anti-phospho-S909/S871 LATS1/2 (9157), and 20

rabbit monoclonal anti-phospho-T1079/T1041 LATS1/2 (D57D3) (8654) (Cell 21

Signaling); and D-luciferin (Summit Pharmaceuticals International). 22

Cell cultures and transfection. HEK293FT and ARPE-19 cells were 23

cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine 24

serum (FBS) and 10 mM Hepes-NaOH at pH 7.4 under 5% CO2 at 37ºC. 25

Human MM IM-9 and human chronic myeloid leukemia K562 cells were cultured 26

in RPMI1640 medium supplemented with 10% FBS and 10 mM Hepes-NaOH 27

pH7.4. HEK293FT cells were purchased from Invitrogen. ARPE-19 and IM-9 28

cells were obtained from Department of Ophthalmology and Department of 29

Hematology, Tokyo Medical and Dental University, respectively. Cell 30

authentication was performed in 2016 using the short tandem repeat analysis by 31

JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and 32




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Nutrition). Mycoplasma contamination was tested by use of e-Myco 1

Mycoplasma PCR Detection Kit (iNtRON Biotechnology). DNA transfection 2

was performed using Lipofectamine 2000 (Invitrogen) and polyethylenemine 3

“Max” (Polysciences). ARPE-19 reporter cells and control reporter cells were 4

prepared using pLL3.7 K122 5

FH-YAP1-TEAD-responsive-promoter-H2B-mCherry and pLL3.7 K122 6

control-TEAD-responsive-promoter-H2B-mCherry. HEK293FT cells expressing 7

mCherry-Halo-YAP1 was generated by use of the retrovirus vector, 8

pQCXIP-mCherry-Halo-YAP1, and puromycin. YAP1-depleted and 9

luciferase-expressing IM-9 cells were prepared by use of 10

pLL3.7-GFP2A-shYAP2 and pLL3.7-H2B-mCherry-2A-luciferase, respectively. 11

Chemical library screening. For the first screening, we used 18,606 12

chemical compounds owned by the Tokyo Medical and Dental University 13

Chemical Biology Screening Center. ARPE-19 reporter cells were plated at 14

3.5x103 cells/well in a 96-well plate. Each plate contained 90 l medium. 24 h 15

later, each compound was applied to the cells to a final concentration of 10 M. 16

The original stock, whose concentration was 10 mM in DMSO, was diluted at 17

1:100 by the medium and 10 l of the diluted sample was added to each plate. 18

As a negative control, DMSO was similarly diluted and added to the cells. 72 h 19

later, the cells were washed with PBS and fixed with 4% formaldehyde/PBS for 20

15 min. Then, the formaldehyde/PBS was changed to PBS containing 1 g/l 21

Hoechst 33342 to visualize the nuclei. Green and red fluorescence intensities 22

of 200 cells in each well were measured by use of ArrayScan VTI (Thermo 23

Fisher Scientific). The ratio of mCherry intensity over GFP intensity was 24

calculated by using BioApplication Compartment Analysis V4 (Thermo Fisher 25

Scientific). The formula for Z-score calculations is as follows: Z -score = (the 26

result obtained for each cell - the median result obtained for each assay plate) / 27

normalized interquartile range (NIQR, namely 0.7413 x (the result corresponding 28

to the first 25 % when ranked in order-the result - the result corresponding to the 29

first 75% when ranked in order) of each assay plate. Each assay plate 30

contained 80 samples. In the first screening, we selected 124 compounds that 31




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gave Z-scores larger than 3.5 as positive samples. We subsequently applied 1

124 compounds to ARPE-19 cells and reanalyzed them. In the second 2

screening, we used the same experimental method as in the first screening but 3

compared mCherry/GFP ratio of each sample with that of the DMSO-treated 4

control sample to select 47 compounds that augmented mCherry/GFP ratio by 5

more than 1.5-fold as positive samples. In the third screening, we evaluated 6

the effects of 47 compounds on YAP1-depedent TEAD reporter assay in 7

HEK293FT cells and YAP1 phosphorylation in ARPE-19 reporter cells. The 8

overview of the screenings is shown in Suppl. Fig. 1. 9

Quantitative reverse transcription-PCR (qRT-PCR). qRT-PCR 10

analysis was performed using SYBR Green (Roche) and ABI7500 Real-Time 11

PCR system (Applied Biosystems). The used primers are as follows; 12

5’-cagcacagcaaattctccaa-3’ and 5’-tggattttgagtcccaccat-3’ for YAP1; 13

5’-ggcaggggagagtgatacaga-3’ and 5’-gaagccaattctcacgaaggg-3’ for MDM2; 14

5’-gggcatctggaccctcctac-3’ and 5’-tcctttcacctggaggacag-3’ for FAS; 15

5’-atgttttctgacggcaacttc-3’ and 5’-atcagttccggcaccttg-3’ for BAX; 16

5’-gtccttcgtgtgggctacat-3’ and 5’-cgaggatcttcggttgacat-3’ for LATS1; 17

5’-ttcatccaccgagacatcaa-3’ and 5’-ctccatgctgtcctgtctga-3’ for LATS2; 18

5’-ccaatgacaacgcctcctg-3’ and 5’-tggtgcagccagaaagctc-3’ for CTGF; 19

5’-agcctcgcatcctatacaacc-3’ and 5’-ttctttcacaaggcggcactc-3’ for CYR61; and 20

5’-ccactcctccacctttgac-3’ and 5’-accctgttgctgtagcca-3’ for GAPDH. 21

RNA interferences. RNA interferences were performed by use of 22

LipofectamineTM RNAiMAX (Life technologies). The double-strand RNAs 23

(dsRNAs) used are: human YAP1#1, s20366, human YAP1#2, s20367, human 24

LATS1, s17392, human LATS2, s25505, and Silencer Select Negative Control 25

No. 2 siRNA (Ambion). 26

Reporter assay. 4x106 HEK293FT cells and HEK293FT cells 27

expressing mCherry-Halo-YAP1 were plated on the collagen-coated 6-cm dish 28

and transfected 0.6 g 8xGT-IIC-δ51LucII luciferase reporter and 0.6 g pCMV 29

alkaline phosphatase. 24h later, the cells from one dish were replated into 24 30

wells in 24-well plate, treated with 10 M each compound for 24h, and harvested. 31




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The cells were lysed in 200l of the lysis buffer (25 mM Tris-HCl pH7.4, 2 mM 1

DTT, 2 mM EDTA, 4 mM EGTA, 4 mM MgCl2, 0.1%(w/v) Triton X-100, and 1 mg/l 2

(Amidinophenyl)methanesulfonyl fluoride (APMSF). The luciferase activity and 3

the alkaline phosphatase activity were assayed with PicaGene (Toyo Ink) and 4

CDP-Star (Roche) and measured by ARVO MX (Perkin Elmer). The alkaline 5

phosphatase assay was performed in 90 mM glycine buffer at pH10.5 with 0.1 6

mM ZnCl2. 7

Subcellular fractionation. ARPE-19 reporter cells were plated at 8

2x105 cells/6-cm dish. 48 h later, the cells were harvested, washed with 9

ice-cold PBS twice, suspended in 200 l of the extraction buffer (10 mM 10

Hepes-NaOH pH8.0, 1.5 mM MgCl2, 10 mM KCl, 0.34 M sucrose, 10% (v/v) 11

glycerol, 0.05% (v/v) NP40, and 1 mM APMSF), kept on ice for 5 min, and 12

suspended up-down with 200 l yellow tip five times. 66.6 l suspension was 13

saved as the total lysate. The remaining sample was centrifuged at 800 x g for 14

5 min at 4ºC. The supernatant was further centrifuged at 20,000 x g for 10 min 15

and the final supernatant was used as the cytosol fraction. The pellet was 16

resuspended in 133.3 l of the extraction buffer and centrifuged again at 800 x g 17

for 4 min. The pellet was resuspended in 133.3 l of the extraction buffer and 18

used as the nuclear fraction. 19

Phosphate-affinity SDS-PAGE. ARPE-19 reporter cells were plated 20

at 1.5x105 cells/well in a 6-well plate and treated with 10 M each compound. 21

24 h later, the cells were harvested and the cell lysates were analyzed by use of 22

Phos-tagTM acrylamide and PolyVinylidene DiFluoride membranes. The 23

signals were measured with ImageJ. 24

LATS kinase assay. 2x106 HEK293FT cells were plated in a 6-cm dish. 25

24 h later the cells were transfected with either pCIneoMyc-LATS1 or 26

pCIneoMyc-LATS2. 24 h after the transfection, the cells were replated at 4x105 27

cells/well in a 12-well plate and treated with 10 M IBS003031. 24 h later, the 28

cells were harvested and the cell lysates were immunoblotted with the indicated 29

antibodies. The sequences around Ser909 of LATS1 and Ser871 of LATS2 and 30

around Thr1079 of LATS1 and Thr1041 of LATS2 are identical and the 31




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phosphorylated LATS1 and LATS2 are detected with the same antibodies. 1

Co-immunoprecipitation and LUMIER assay. HEK293FT cells were 2

plated at 8x105 cells/well in a 6-well plate, were transfected with various 3

combinations of expression vectors. After overnight culture, IBS003031 was 4

added to a final concentration of 10 M. 24 h later, the cells were harvested 5

and lysed in the lysis buffer (25 mM Tris-HCl pH7.4, 150 mM NaCl, 10 mM MgCl2, 6

2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 50 M APMSF, 10 mg/l 7

leupeptin, and 10 mg/l pepstatin A). After centrifugation at 20,000 x g for 10 8

min, the supernatant was collected and the immunoprecipitation was performed 9

with anti-DYKDDDDK-tag beads. For LUMIER assay, HEK293FT cells were 10

plated at 8x105 cells/well in a 6-well plate and were transfected with various 11

combinations of the expression vectors. 24 h later, the cells from each well 12

were replated into 6 wells of a 24-well plate and treated with DMSO or 10 M 13

IBS003031 for 24 h. The immunoprecipitation was performed with 14

anti-DYKDDDDK tag beads. The immunoprecipitates were washed vigorously 15

and the luciferase activity was measured as described for the reporter assay. 16

Cell proliferation assay. The colorimetric MTT assay was performed 17

with thiazolyl blue tetrazolium bromide. The insoluble formazan was dissolved 18

with 50 l DMSO and the absorbance at 570 nm was measured with SmartSpec 19

3000 (Bio-Rad). 20

Lactate dehydrogenase (LDH) assay. K562 cells were plated at 21

3x106 cells/dish in a 6-cm dish and transfected with control siRNA or YAP1#1 22

siRNA. 48 h later, the cells were re-plated at 3x104 cells/well in a 96-well plate. 23

The cells were treated with either control DMSO or 10 M IBS003031 for 48 h. 24

As K562 cells, although hematopoietic cells, attached to the plate, 50 l cell-free 25

medium was collected and LDH activity was assayed using 26

2-p-iodophenyl-3-p-nitrophenyl tetrazolium chloride, N-methylphenazonium 27

methyl sulfate, nicotinamide adenine nucleotide, and lactic acid(37). All these 28

reagents were obtained form Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 29

Detection of sub-G1 population. IM-9 cells were harvested and fixed 30

in ice-cold 70% (v/v) ethanol, washed with PBS, and resuspended in PBS 31




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containing 10 mg/l propidium iodide and 1 g/l RNaseA. The sub-G1 population 1

was evaluated with FACS Calibur (BD Biosciences). The data were analyzed 2

with BD CellQuest Pro Software. 3

Animal experiments. All animal experiments were approved by the 4

Tokyo Medical and Dental University Animal Care and Use Committee. A total 5

of 2x106 IM-9 cells carrying luciferase were injected intraperitoneally into female 6

C.B17/Icr-scidJcl mice (5 weeks old) (Clea Japan. Inc.). Tumor progression 7

was monitored by bioluminescence imaging on IVIS Lumina system 8

(PerkinElmer). Control DMSO or IBS003031 (5 mg/kg body weight) was 9

intraperitoneally daily. To evaluate the effect of IBS003031 on YAP1 in vivo, 10

3.5x106 IM-9 cells were subcutaneously transplanted with Matrigel (BD 11

Biosciences). 28 days later, after tumor formation, DMSO or IBS003031 (5 mg/ 12

kg body weight) was intraperitoneally injected. One day later, mice were 13

sacrificed and tumors were excised. Tumors were fixed with 4% (w/v) 14

formaldehyde in PBS for overnight and dehydrated in 70% ethanol for another 15

overnight. After embedded in paraffin blocks and sliced into 4 m slices, the 16

samples were deparaffined and heated in citrate buffer to activate antigens. 17

After endogenous peroxidase was inactivated, the samples were blocked with 18

3% (w/v) BSA in PBS and immunostained with rabbit monoclonal anti-YAP1 19

antibody. Signals were labeled by using R.T.U. VECTASTAIN kit (VECTOR 20

LABORATRIES Inc.) and SIGMAFASTTM 3,3’-diaminobenzidine tablets. 21

Statistical analysis. Statistical analyses were performed with 22

student’s t test for the comparison between two samples and analysis of 23

variance with Dunnett’s test for the multiple comparison using the GraphPad 24

Prism 5.0 (GraphPad Software). 25

26

Results 27

28

The generation of ARPE-19 cells stably expressing the TEAD-responsive 29

reporter. 30

YAP1 interacts with various transcription factors including TEAD, SMAD, Runx2, 31

and p73. In the in vitro reporter assays, YAP1 enhances the reporter activities 32




13 / 32

that respond to these transcription factors. However, the recent study using 1

ChIP-seq demonstrates that TEAD is associated with 75% of YAP1 peaks and 2

that TEAD is the most prominent partner of YAP1(38). Moreover, the TEAD 3

reporter assay exhibits the most remarkable activity and is the easiest to monitor. 4

We expressed mCherry-fused histone2B (H2B-mCherry) under the 5

TEAD-responsive promoter and FLAG-tagged YAP1 linked to the internal 6

ribosome entry site (IRES) and GFP under the cytomegalovirus promoter (CMV) 7

in immortalized human retinal pigment epithelial ARPE-19 cells (Fig. 1A). We 8

evaluated mCherry signals driven by the TEAD-responsive promoter. YAP1 9

knockdown suppressed mCherry signals, supporting that mCherry signals reflect 10

YAP1 activity and that the ARPE-19 reporter cells can be used for the screening 11

of YAP1 activators (Fig. 1B). 12

13

The identification of the compounds that enhance the H2B-mCherry 14

expression. 15

We applied 18,606 chemical compounds to ARPE-19 reporter cells and cultured 16

them for 72 h to select the compounds that changed the expression of 17

H2B-mCherry (Suppl. Fig. 1). We obtained 124 putative enhancers in the 18

initial screening, which gave Z-scores higher than 3.5 (Fig. 1C). For the 19

second selection, we directly compared these compounds with the control and 20

selected 47 compounds that augmented the signals by more than 1.5-fold. To 21

confirm that the compounds enhanced the signal via YAP1, we applied the 22

compounds to ARPE-19 control reporter cells expressing only the 23

TEAD-responsive reporter. The effects of the compounds were remarkably 24

attenuated, supporting that the exogenously expressed YAP1 is required for the 25

compounds to enhance H2B-mCherry signal (Suppl. Fig. 2A). Considering the 26

risk that the concentration employed might be too high, we applied 27

representative enhancers at the lower dose. IBS003031 up-regulated 28

H2B-mCherry signals at 1 M and 3 M, whereas IBS012851 was active at 3M 29

(Suppl. Fig. 2B). Moreover, when YAP1 was knocked down in the reporter 30

cells, the effects of the compounds were abolished (Fig. 1D). These findings 31




14 / 32

corroborate that the compounds enhance H2B-mCherry signal via YAP1. 1

2

The effect on the TEAD reporter activity. 3

We next confirmed the effects of these candidate compounds on the 4

conventional TEAD-responsive reporter activity in HEK293FT cells. We 5

prepared HEK293FT cells stably expressing YAP1 and after the confirmation 6

that YAP1 overexpression did not show any significant effect on the cell viability 7

(data not shown), performed the reporter assay. 6 compounds (IBS003031, 8

IBS012851, CBI001316, IBS002470, IBS007607, and IBS014765) enhanced 9

the reporter activity more than 1.75-fold (Fig. 2A, black columns; Fig. 2B, 10

black dots). We designated these compounds as TEAD reporter enhancers 11

(TRE). 6 compounds (IBS011345, IBS006077, IBS011342, IBS006808, 12

IBS004557, and IBS001781) rather suppressed the reporter activity (Fig. 2A, 13

white columns; Fig. 2B, white dots). We speculate that the discrepancy 14

between the ARPE-19-based assay and the conventional reporter assay might 15

be caused by the differences of cells and the duration of the treatment of the 16

compounds. 17

18

The effects on the phosphorylation of YAP1. 19

YAP1 is negatively regulated by the canonical Hippo pathway via the 20

phosphorylation(8). To directly address whether and how compounds affect the 21

phosphorylation of YAP1, we treated APRE-19 reporter cells with the 22

compounds and ran the cell lysates on the Phos-tag SDS-PAGE. The lowest 23

bands were detected with anti-FLAG antibody but not with anti-phospho-S127 24

YAP1 antibody (Suppl. Fig. 3A). The upper three bands were detected with 25

both the antibodies. Moreover, LATS1/2 silencing increased the lowest band 26

(Suppl. Fig. 3A, an arrowhead). These findings support that the lowest band 27

corresponds to the unphosphorylated YAP1. We quantified signals by use of 28

ImageJ and calculated the relative amount of the unphosphorylated YAP1. 29

Among 6 TRE compounds (Fig. 3A and 3B, arrows), IBS014765 did not 30

increase the unphosphorylated YAP1 (Fig. 3A and 3B, stars). Other 5 TRE 31

compounds did increase unphosphorylated YAP1 (Fig. 3A and 3B). 32




15 / 32

IBS003031 and IBS012851 remarkably decreased the phosphorylated YAP1 1

signal (Fig.3A, white arrowheads). These compounds were speculated to 2

activate YAP1 via the canonical Hippo pathway, which increases phosphorylated 3

YAP1 and decreases unphosphorylated YAP1. Interestingly, the structures of 4

IBS003031 and IBS012851 are similar (Fig. 3C). We have recently reported 5

that ethacridine, which was originally identified as a TAZ activator, reduces 6

phosphorylated YAP1 and increases unphosphorylated YAP1(30). Ethacridine 7

also has an acridine backbone. We speculate that these compounds activate 8

YAP1 through the common target molecule. After we confirmed that 9

IBS003031 enhanced the expression of YAP1 target genes, CTGF and CYR61, 10

in U2OS cells, we decided to focus on IBS003031 (Suppl. Fig. 4). 11

12

The effect of IBS003031 on LATS1/2 activities 13

We postulated that IBS003031 might activate YAP1 through the inhibition of the 14

phosphorylation by LATS kinases or the facilitation of the dephosphorylation. 15

We first tested whether IBS003031 could inhibit LATS1/2 activities. We 16

expressed Myc-LATS1 and Myc-LATS2 in HEK293FT cells and treated the cells 17

with IBS003031. The phosphorylation at S909/S871 and T1079/T1041 of 18

LATS1/2 is essential for the activities of LATS1/2(39). We immunoblotted the 19

cells lysates with the antibodies against phosphorylated-S909/S871 and 20

-T1079/T1041 of LATS1/2. IBS003031 increased the expression of 21

Myc-LATS1 and augmented the phosphorylation at S909 and T1079 (Fig. 4A, 22

the left panel). IBS003031 slightly increased the expression of Myc-LATS2, 23

and enhanced the phosphorylation at S871 and T1041 (Fig. 4A, the right 24

panel). We next tested whether IBS003031 blocked the binding of YAP1 to 25

LATS1/2. We expressed luciferase-fused YAP1 with FLAG-LATS1 and 26

LATS2-FLAG, conducted the immunoprecipitation with anti-DYKDDDDK-tag 27

antibody beads, and measured the co-immunoprecipitated luciferase activity 28

(Fig. 4B). Consistent with the result in Figure 3A, IBS003031 enhanced the 29

expression of FLAG-LATS1 and LATS2-FLAG, and consequently increased the 30

amount of FLAG-LATS1 and LATS2-FLAG in the immunoprecipitates (Fig. 4B, 31

the bottom immunoblottings). Luc-YAP1 that was co-immunoprecipitated 32




16 / 32

with FLAG-LATS1 and LATS2-FLAG was also higher under the treatment with 1

IBS003031. Based on these findings, we conclude that it is unlikely that 2

IBS003031 decreases YAP1 phosphorylation through inhibiting the activation of 3

LATS kinases or blocking the interaction of YAP1 and LATS kinases. 4

5

The effect of IBS003031 on the dephosphorylation of YAP1. 6

We next hypothesized that IBS003031 might promote the dephosphorylation of 7

YAP1. We treated ARPE-19 reporter cells expressing FLAG-YAP1 with 8

IBS003031 and analyzed the cell lysates on the Phos-tag SDS-PAGE (Fig. 5A). 9

Under the treatment of IBS003031, phosphorylated YAP1 decreased and 10

concomitantly unphosphorylated YAP1 increased in a time-dependent manner. 11

We also performed the subcellular fractionation. IBS003031 increased 12

unphosphorylated YAP1 in both the cytoplasm and the nucleus, supporting that 13

YAP1 is dephosphorylated in the cytoplasm and then transported into the 14

nucleus (Fig. 5B). 15

16

The effect of IBS003031 on the interaction of YAP1 with protein 17

phosphatase (PP) 1A and PP2A. 18

PP1A and PP2A are involved in the dephosphorylation of YAP1(40,41). We 19

examined whether IBS003031 could strengthen the interaction of YAP1 with 20

PP1A and PP2A. We expressed luciferase-fused PP1A and PP2A with 21

FLAG-YAP1 in HEK293FT cells and measured the luciferase activity 22

co-immunoprecipitated with FLAG-YAP1. IBS003031 slightly enhanced the 23

interaction of YAP1 with PP1A but the difference was not significant, while it 24

significantly increased the interaction between YAP1 and PP2A (Fig. 5C). We 25

further confirmed that IBS003031 enhanced the interaction between YAP1 and 26

PP2A in U2OS cells (Suppl. Fig. 5). In conclusion, IBS003031 is supposed not 27

to suppress the phosphorylation of YAP1 by LATS kinases but is likely to 28

promote the dephosphorylation of YAP1. 29

30

The effect of IBS003031 on MM cells in vitro. 31

YAP1 induces apoptosis in MM cells(19). In normal cells, upon DNA damage, 32




17 / 32

ABL1 is released from 14-3-3 and enters the nucleus(42). The nuclear ABL1 1

phosphorylates YAP1 at tyrosine, enhances the association of YAP1 with p73, 2

and up-regulates p73-dependent pro-apoptotic gene transcription(18). In MM 3

cells, due to the low expression of YAP1, ABL1-YAP1-p73 axis is 4

compromised(19). Certain MM cells express phosphorylated H2A.X termed 5

as -H2A.X, which is a hallmark of DNA damage response, but escape apoptosis 6

because of the low expression of YAP1(43). However, when YAP1 is 7

exogenously expressed or YAP1 is activated by the suppression of the Hippo 8

pathway, MM cells begin to die. We presumed that YAP1 activators would 9

trigger apoptosis in MM cells. In this study, we used IM-9 cells and treated 10

them with various doses of IBS003031 and performed MTT assay. IBS003031 11

decreased the viability in the dose-dependent manner (Fig. 6A, the left). IM-9 12

cells expressed-H2A.X with no treatment, which means that IM-9 cells show 13

DNA damage response at the basal condition (Fig. 6A, 0 M in the immunoblot 14

for -H2A.X). IBS003031 enhanced Bax expression in a dose-dependent 15

manner (Fig. 6A, the immunoblot for Bax). The treatment with 6 M and 9 16

M IBS003031 apparently decreased -H2A.X expression, but as -tubulin 17

expression was also reduced, we speculated that the decrease of -H2A.X 18

expression might be due to the decrease of the total cell number. We also 19

confirmed in FACS analysis that IBS003031 increased sub-G1 population in a 20

dose-dependent manner (Fig. 6B, the upper panels). YAP1 depletion 21

reduced the IBS003031-induced sub-G1 population (Fig. 6B, the lower panels). 22

Trypan blue exclusion assay further supported that IBS003031 caused cell death 23

more strongly in parent IM-9 cells than in YAP1-depleted IM-9 cells (Fig. 6C, the 24

left panel). In the immunoblotting, IBS003031-induced Bax expression was 25

attenuated by YAP1 depletion (Fig. 6C, the right panel). Imatinib, ABL1 26

inhibitor, reduced the effect of IBS003031 in MTT assay (Fig. 6D). All these 27

findings support that IBS003031 induces apoptosis in IM-9 cells through ABL1 28

and YAP1. We also observed that IBS003031 suppressed cell viability and 29

induced cell death in human myeloid leukemia K562 cells depending on YAP1 30

(Suppl Fig. 6). 31




18 / 32

1

The effect of IBS003031 on p73-dependent transcription. 2

IBS003031 increased the amount of MDM2, FAS, and BAX in mRNA level, 3

which are p73 target genes (Fig. 7A). Accordingly, in HEK293FT cells, 4

IBS003031 stimulated MDM2-promoter luciferase reporter activity and YAP1 5

knockdown antagonized the effect of IBS003031 (Fig. 7B). Moreover, 6

IB003031 enhanced the coimmunoprecipitation of FLAG-p73 and GFP-YAP1 7

from HEK293FT cells (Fig. 7C). 8

9

The effect of IBS003031 on MM cells in vivo. 10

We transplanted luciferase-expressing IM-9 cells into the immunocompromised 11

mice. 20 days after transplantation, control DMSO or IB003031 (a final 12

concentration of 5 mg/kg body weight) was injected intraperitoneally daily for 13

one week (Fig. 8A). Tumor growth was evaluated in the bioluminescence 14

imaging (Fig. 8B). The trend of the suppression of the growth by IBS003031 15

was observed (Fig. 8C). In the parallel experiment, we subcutaneously 16

transplanted IM-9 cells and after tumor was formed, DMSO or IBS003031 was 17

injected intraperitoneally (Suppl. Fig. 7A). One day later, tumor was isolated 18

and endogenous YAP1 was immunostained. IBS003031 indeed increased 19

nuclear YAP1 (Suppl. Fig. 7B). 20

21

Discussion 22

In this study, we expressed YAP1 and TEAD-responsive fluorescent protein 23

reporter in human retinal pigment ARPE-19 cells and used them to detect small 24

chemical compounds that enhance the reporter activity (Fig. 1). ARPE-19 cells 25

are immortalized cells, but show contact inhibition, suggesting that the Hippo 26

pathway is intact. In the initial screening by using 18,606 compounds, we 27

tested 80 compounds as one set in a 96-well plate and calculated Z-score in 28

each set. The first screening gave 124 compounds with Z-score higher than 29

3.5 (Fig. 2). In the second screening, we directly compared them with the 30

control and further selected 47 compounds that enhanced the reporter by more 31

than 1.5. These compounds did not enhance the fluorescence reporter in 32




19 / 32

ARPE-19 cells expressing only TEAD-responsive reporter, supporting that the 1

compounds up-regulated the reporter via exogenously expressed YAP1. In the 2

conventional reporter assay using HEK293FT cells and TEAD-responsive 3

luciferase reporter, 6 compounds attenuated the reporter activity (Fig. 2). 4

ARPE-19 reporter cells were exposed to the compounds for 72 h, whereas 5

HEK293FT cells were treated with the compounds for 18 h. The reason why 6 6

compounds show the unexpected effect may be the difference of the 7

experimental condition. 8

The phosphorylation by LATS kinases is best characterized and the 9

most prominent regulatory mechanism of YAP1. According to the canonical 10

Hippo pathway, YAP1 activators are predicted to decrease YAP1 11

phosphorylation and increase the amount of unphosphorylated YAP1. Among 12

6 compounds that remarkably enhanced the reporter activity in the conventional 13

reporter assay, IBS003031 and IBS012851, faithfully to the canonical Hippo 14

pathway, increased the amount of unphosphorylated YAP1 and decreased that 15

of phosphorylated YAP1 (Fig. 4). Intriguingly, the structures of both 16

compounds and ethacridine that we previously reported as a YAP1/TAZ activator 17

are similar. We speculate that these compounds target the same or the related 18

molecule. 19

In this study, we wanted to conclude whether bona fide YAP1 activators 20

can be obtained through the screening with ARPE-19 reporter cells. To 21

facilitate the analysis, we focused on IBS003031 that remarkably increased the 22

amount of unphosphorylated YAP1 and decreased that of phosphorylated YAP1. 23

IBS003031 enhanced the expression of LATS kinases. IBS003031 did not 24

inhibit the activities of LATS kinases and did not block the interaction between 25

YAP1 and LATS kinases (Fig. 3). We could conclude that the 26

IBS003031-induced up-regulation of unphosphorylated YAP1 is not caused by 27

the inhibition of the phosphorylation. On the other hand, IBS003031 enhanced 28

the interaction of YAP1 with protein phosphatases and induced the 29

dephosphorylation of YAP1 (Fig. 4 and Suppl. Fig. 5). The subcellular 30

fractionation experiment revealed that unphosphorylated YAP1 increased not 31

only in the nucleus but also in the cytoplasm under the treatment with 32




20 / 32

IBS003031. This finding suggests that the import of unphosphorylated YAP1 1

into the nucleus is limited and that a slight increase in the nuclear 2

unphosphorylated YAP1 may be sufficient to up-regulate gene transcription. 3

The precise mechanism how IBS003031 enhances the interaction between 4

YAP1 and protein phosphatases is not clear. Apoptosis-stimulating of p53 5

protein 2 functions as a scaffold for YAP1 and PP1A to promote the interaction of 6

both proteins(40). -Catenin inhibits the interaction between YAP1 and 7

PP2A(41). Proteomic studies have revealed that the protein network 8

underlying the Hippo pathway includes several interactions with serine and 9

threonine protein phosphatases(44). IBS003031 may work through these 10

interactions. 11

YAP1 is primarily thought to be an oncoprotein(9). The expression of 12

nuclear YAP1 correlates with poor prognosis in cancer patients. Numerous 13

reports demonstrate that YAP1 activation leads to oncogenesis. Meanwhile, 14

YAP1 is implicated in p73-dependent transcription of pro-apoptotic genes(17). 15

YAP1 depletion suppresses cell death and promotes tumor formation in certain 16

cancers. ABL1 is important for YAP1 to adopt the fate as a tumor 17

suppressor(18). DNA damage induces the nuclear localization of ABL1, which 18

phosphorylates YAP1. Tyrosine-phosphorylated YAP1 interacts with p73 and 19

up-regulates p73-dependent transcription. A recent study has revealed the 20

tumor suppressive aspect of YAP1 in MM cells(19). MM cells, in which YAP1 21

expression is low, do not undergo apoptosis in response to DNA damage, but 22

begin to die when YAP1 is exogenously expressed or up-regulated by the 23

suppression of mammalian Ste20-like kinase 1. Based on this report, we 24

speculated that YAP1 activator could induce cell death in MM cells. As 25

expected, IBS003031 triggers cell death in human MM IM-9 cells (Fig. 5). 26

YAP1 depletion and ABL1 inhibitor blocked this effect. IBS003031 27

strengthened the interaction between YAP1 and p73 (Fig. 6). All these findings 28

indicate that IBS003031 induces apoptosis in IM-9 cells via ABL1-YAP1-p73 axis. 29

We further confirmed that IBS003031 suppressed the growth of IM-9 cells in vivo 30

and increased the nuclear YAP1 (Fig. 7 and Suppl. Fig. 7). The importance of 31

ABL1-YAP1-p73 axis as a barrier against hematological malignancies was 32




21 / 32

discussed in the previous paper(19). We found that IBS003031 induced cell 1

death in human myeloid K562 cells via YAP1. Therefore, YAP1 activation may 2

be useful to control not only MM cells but also other blood cancer cells (Suppl. 3

Fig. 6). The study including animal experiments will be awaiting. 4

In conclusion, the compound screening by use of ARPE-19 reporter cells 5

provided us with a novel YAP1 activator. We have also demonstrated the 6

possibility that YAP1 activation can be a choice in the treatment of MM cells. 7

8

Acknowledgements 9

We acknowledge Hideyuki Saya, Tadashi Yamamoto, Sumiko Watanabe, and 10

Hiroshi Sasaki for materials. 11

12

Founding information 13

This work was supported by research grants from Japan Society for the 14

Promotion of Science (JSPS) (26460359, 26293061), and the Mitsubishi 15

Foundation. 16

17

Disclosure Statement 18

The authors dictate no conflict of interest. 19

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Legends to Figures 13

14

Figure 1. (A) Schematic of the reporter construct used in this study. 15

FLAG-His6-YAP1 (FH-YAP1) gene followed by internal ribosomal entry sites 16

(IRES) and GFP gene are cloned under CMV promoter. Histone 2B-mCherry 17

(H2B-mCherry) gene is regulated under the TEAD-responsive element 18

harboring eight repeats of gacacacattccacagctg. (B) H2B-mCherry and GFP in 19

ARPE-19 cells. H2B-mCherry and GFP are visible at the basal condition. 20

When YAP1 is knocked down, GFP expression is suppressed, because GFP 21

gene is linked to YAP1 gene by IRES sequence. At the same time, 22

H2B-mCherry signal is also reduced. H2B-mCherry signal was measured by 23

ArrayScan VTI in 100 cells and demonstrated as the histograms. YAP1 24

knockdown shifted the distribution of H2B-mCherry signals to the leftward. 25

Bars, 200m. (C) 18,606 compounds were applied to the reporter cells in 26

96-well plates and H2B-mCherry signals were measured in 3,500 cells for each 27

compound. Z-scores were calculated among the compounds in each plate, in 28

which 80 compounds were tested. (D) The representative image of the 29

ARPE-19 reporter cells treated with YAP1 activator. When the reporter cells 30

were exposed to 10 M IBS003031, H2B-mCherry signals were enhanced, but 31




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YAP1 knockdown canceled the effect of the compound. Bars, 100m. 1

2

Figure 2. The effect of the compounds on YAP1-depedent TEAD-responsive 3

reporter activity in HEK293FT cells. 8xGT-IIC-δ51LucII luciferase reporter 4

(0.6g) and pCMV alkaline phosphatase (0.6g) were transfected into 4x106 5

HEK293FT cells stably expressing YAP1 in a 6-cm dish. 24 h later, the cells 6

were replated at 2x105 cells/well in a 24-well plate. The cells were cultured with 7

10 M each compound for 18 h. The luciferase activity was determined and 8

calibrated by alkaline phosphatase activity. (A) Black columns show the 9

compounds (IBS003031, IBS012851, CBI001316, IBS002470, IBS007607, and 10

IBS014765) that enhanced the activity more than 1.75-fold, while white columns 11

demonstrate the compounds that suppressed the activity. Other compounds 12

were depicted as gray bars. (B) The results of the reporter assay are shown in 13

descending order of magnitude. Black dots represent the compounds that 14

enhance the reporter activity more than 1.75-fold. 15

16

Figure 3. The effect of the compounds on YAP1 phosphorylation. ARPE-19 17

reporter cells were plated at 1.5x105 cells/well in a 6-well plate and treated with 18

10 M each compound for 24 h. The cell lysates were analyzed on Phos-tag 19

gels and immunoblotted with anti-FLAG-antibody. The samples were also run 20

on the conventional SDS-PAGE gels and immunoblotted with anti-FLAG and 21

anti--actin antibodies. The lysates of LATS1/2-depleted cells were charged on 22

the last lane in each gel, in which the lowest band (unphosphorylated YAP1) 23

increased (arrowheads). Six compounds (IBS003031, IBS012851, CBI001316, 24

IBS002470, IBS007607, and IBS014765) that enhanced the TEAD reporter 25

activity were indicated with arrows. IBS003031, IBS012851, CBI001316, 26

IBS002470, and IBS007607 increased unphosphorylated YAP1, while 27

IBS014765 did not increase the unphosphorylated YAP1 (a star). IBS003031 28

and IBS012851 reduced YAP1 phosphorylation (white arrowheads). 29

(B) The signals in Phos-tag gels were measured with ImageJ. The relative 30

intensity of the lowest band over the sum of all the bands were calculated for 31




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each compound. Arrows indicate 6 compounds that enhanced the 1

TEAD-responsive reporter activity in Figure 2A. (C) Structures of IBS003031, 2

IBS012851, and ethacridine. 3

4

Figure 4. The effect of IBS003031 on the phosphorylation at S909/S971 and 5

T1079/T1041 of LATS1/2. (A) HEK293FT cells were plated at 2x106 cells/6-cm 6

dish and transfected with pCIneoMyc-LATS1 (5 g) or pCIneoMyc-LATS2 (5 g). 7

The cells were cultured overnight, replated at 4x105 cells/well in a 12-well plate, 8

and then treated with either DMSO or 10 M IBS003031 for 24 h. The cell 9

lysates were immunoblotted with anti-Myc antibody and the phospho-specific 10

antibodies against phosphorylated S809/S871 and T1079/T1041 of LATS1/2. 11

The immunoblotting with anti--tubulin antibody was shown as a loading control. 12

IBS003031 increased Myc-LATS1 expression. (B) HEK293FT cells were 13

plated at 8x105 cells/well in a 6-well plate, transfected with pCIneoLuc-YAP1 (1 14

g), pCIneoFLAG-LATS1 (1 g), or pcDNA-LATS2-FLAG (1 g) and cultured 15

overnight. The cells were replated at 1.3x105 in a 24-well plate and culture for 16

24 h with DMSO (the second column (Control)) or 10 M IBS003031. 17

FLAG-LATS1 or LATS2-FLAG was immunoprecipitated. The luciferase activity 18

co-immunoprecipitated was measured. The first column represents the 19

luciferase activity in the immunoprecipitate from the cells, in which Luc-YAP1 20

was expressed with GFP instead of FLAG-LATS1 or LATS2-FLAG. The 21

immunoblotting at the bottom shows that IBS003031 remarkably increased 22

FLAG-LATS1 in the immunoprecipitate, which may reflect the enhanced 23

expression of LATS1 in the presence of IBS003031 shown in Figure 3A. 24

LATS2-FLAG in the immunoprecipitate was also slightly increased by 25

IBS003031. The data are mean +/- SD. ***p<0.001. 26

27

Figure 5. The effect of IBS003031 on the dephosphorylation of YAP1 and the 28

interaction between YAP1 and protein phosphatases. In (A), ARPE-19 29

reporter cells were plated at 1.5x105 cells/well in a 6-well plate. In (B), 2x105 30

cells were plated in a 6-cm dish. The cells were treated with DMSO or 10 M 31




30 / 32

IBS003031 for the indicated periods of time in (A) and for 24 h in (B). In (A), 1

the whole cell lysates were analyzed on Phos-tag gels and immunoblotted with 2

anti-FLAG antibody. The samples were also run on the conventional 3

SDS-PAGE and immunoblotted with anti-FLAG and anti--actin antibodies to 4

demonstrate that all lanes contain almost the same amount of proteins. Under 5

the treatment with IBS003031, the unphosphorylated YAP1 (an arrow) 6

appeared at 3 h and the phosphorylated YAP1 gradually decreased. In (B), 7

the subcellular fractionation was performed to separate the nuclear and the 8

cytoplasmic fractions. IBS003031 increased the amount of unphosphorylated 9

YAP1 in the cytoplasmic fraction. Unphosphorylated YAP1 (an arrow) in the 10

nuclear fraction increased. -Tubulin and Poly(ADP-ribose) polymerase 11

(PARP) were used as the cytoplasmic and nuclear markers. (C) 12

Luciferase-fused protein phosphatase 1A and 2A (Luc-PP1A and Luc-PP2A) 13

were co-expressed with GFP or FLAG-YAP1 in HEK293FT cells. The cells 14

were treated with DMSO or 10 M IBS003031 for 24 h and the 15

immunoprecipitation was performed with anti-DYKDDDDK-tag antibody beads. 16

The luciferase activity attached to the beads was measured. The first column 17

represents the luciferase activity that non-specifically bound to the beads. 18

The value of the luciferase activity of the immunoprecipitate from the cells 19

treated with DMSO is set as 1. The data are mean +/- SD. n.s, not 20

significant; and *p<0.05. 21

22

Figure 6. The effect of IBS003031 on human MM IM-9 cells. (A) IM-9 cells 23

were plated at 2x104 cells/well in a 96-well plate. The cells were cultured in the 24

presence of various doses of IBS003031 for 48 h. The cell viability was 25

assessed by MTT assay and the viability of the cells with no treatment of 26

IBS003031 was set as 1. For the immunoblotting, the cells were harvested at 27

24 h and the cell lysates were immunoblotted with the indicated antibodies. (B) 28

Parent and YAP1-depleted IM-9 cells were plated at 2x105 cells in a 6-cm dish 29

and treated with IBS003031 at various doses for 48 h. The cells were fixed in 30

the ice-cold 70% (v/v) ethanol and incubated for 30 min with 1 mg/l propidium 31




31 / 32

iodide and 10 mg/l RNaseA. The sub-G1 population was evaluated with FACS 1

Calibur. The data were analyzed with BD CellQuest Pro Software. YAP1 2

depletion was confirmed by qRT-PCR (the right histogram). (C) Parent and 3

YAP1-depleted IM-9 cells were plated at 1x105 cells/6-cm dish and treated with 4

DMSO or 9 M IBS003031 for 48 h. The viable cells were evaluated in the 5

trypan blue exclusion assay (the left panel). The ratio of trypan blue-positive 6

cells for each condition is indicated. Parent IM-9 cells and YAP1-depleted IM-9 7

cells were treated with IBS003031 at various doses for 48 h and the cell lysates 8

were immunoblotted with anti-Bax antibody (the right panel). ***, p<0.001. 9

(D) IM-9 cells were plated at 1x106 cells/10-cm dish and cultured without or with 10

1M Imatinib overnight. The cells were replated at 2x104 cells/well in a 96-well 11

plate and cultured with IBS003031 at various doses without or with 1 M Imatinib. 12

The cell viability was assessed by MTT assay and the viability of the cells with no 13

treatment of IBS003031 was set as 1. **, p<0.01. 14

15

Figure 7. The effect of IBS003031 on p73-dependent transcription. (A) IM-9 16

cells were plated at 3x105 cells/well in a 12-well plate. 24 h later, IBS003031 17

was added to a final concentration of 9 M. For control cells, DMSO was 18

added. The cells were cultured for 24h. Total RNA was isolated and 19

qRT-PCR was performed. The value for the control cells is set as 1. *, p<0.05; 20

**, p<0.01; and ***, p<0.001. (B) YAP1 was knocked down. 48 h later, 21

HEK293FT cells were transfected with MDM2-promoter luciferase reporter (0.3 22

g), and pCMV alkaline phosphatase (0.3 g). 5 h later, the cells were replated 23

at 3.2x105 cells/well in a 12-well plate and cultured with or without IBS003031 for 24

24 h. The reporter assay was performed as described for Figure 2A. YAP1 25

depletion was confirmed by the immunoblotting (the bottom). ***, p<0.001. 26

(C) GFP-YAP1 and FLAG-p73 were expressed in HEK293FT cells. The cells 27

were treated with 10 M IBS003031. FLAG-p73 was immunoprecipitated. 28

The co-immunoprecipitated GFP-YAP1 was increased by IBS003031 treatment. 29

30

Figure 8. The effect of IBS003031 on the proliferation of IM-9 cells in mice. 31




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2x106 IM-9 cells or YAP1-depleted IM-9 cells carrying luciferase were injected 1

into C.B17/Icr-scidJcl mice intraperitoneally. 20 days later, control DMSO or 2

IBS003031 (5 mg/kg body weight) was injected intraperitoneally daily for 7 days. 3

(A) Design of the experiment. (B) Representative images from 4

bioluminescence imaging of mice carrying IM-9 cells. (C) Distribution of the 5

fold increase of the bioluminescence signal intensity at day 7. 6

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Published OnlineFirst October 23, 2017.Mol Cancer Res Junichi Maruyama, Kazutoshi Inami, Fumiyoshi Michishita, et al. Functional Screen, Limits Multiple Myeloma GrowthNovel YAP1 Activator, Identified by Transcription-based

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