YES1 Is a Targetable Oncogene in Cancers Harboring YES1 Gene Amplification

Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 YES1 is a targetable oncogene in cancers harboring YES1 gene

2 amplification

3 Natsuki Hamanaka1*, Yoshito Nakanishi1*, Takakazu Mizuno1, Kana Horiguchi-Takei1,

4 Nukinori Akiyama1, Hiromi Tanimura1, Masami Hasegawa1, Yasuko Satoh1, Yukako

5 Tachibana1, Toshihiko Fujii1, Kiyoaki Sakata1, Kiyomoto Ogasawara1, Hirosato Ebiike1,

6 Hiroshi Koyano1, Haruhiko Sato1, Nobuya Ishii1 and Toshiyuki Mio1

7 Affiliations

8 1Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa

9 247-8530, Japan

10 * Co-first author

11 Running Title:

12 Effective YES1-YAP1 blockade on YES1 gene–amplified cancers

13 Keywords: YES1, YAP1, Oncogene addiction, Gene amplification, Tyrosine kinase inhibitor

14 Corresponding Author

15 Yoshito Nakanishi, Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara,

16 Kamakura, Kanagawa, 247-8530 Japan. Phone: 81-70-4314-8769; Fax: 81-467-47-6561;

17 E-mail: [email protected]

18 Disclosure of potential conflict of interest

19 All authors are employees of Chugai Pharmaceutical Co., Ltd. The authors declare no

20 potential conflicts of interest.

21 Financial Support

22 This study was funded by Chugai Pharmaceutical Co., Ltd.

23 Word count: 4885

24 Total number of figures and tables: 7

25 Abstract

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Targeting genetic alterations of oncogenes by molecular targeted agents (MTAs) is

2 an effective approach for treating cancer. However, there are still no clinical MTA options for

3 many cancers, including esophageal cancer. We used a shRNA library to screen for a new

4 oncogene in the esophageal cancer cell line KYSE70 and identified YES proto-oncogene 1

5 (YES1) as having a significant impact on tumor growth. An analysis of clinical samples

6 showed that YES1 gene amplification existed not only in esophageal cancer but also in lung,

7 head and neck, bladder, and other cancers, indicating that YES1 would be an attractive target

8 for a cancer drug. Since there is no effective YES1 inhibitor so far, we generated a YES1

9 kinase inhibitor, CH6953755. YES1 kinase inhibition by CH6953755 led to antitumor

10 activity against YES1-amplified cancers in vitro and in vivo. Yes-associated protein 1 (YAP1)

11 played a role downstream of YES1 and contributed to the growth of YES1-amplified cancers.

12 YES1 regulated YAP1 transcription activity by controlling its nuclear translocation and serine

13 phosphorylation. These findings indicate that the regulation of YAP1 by YES1 plays an

14 important role in YES1-amplified cancers and that CH6953755 has therapeutic potential in

15 such cancers.

17 Statement of Significance

18 Findings identify the SRC family kinase YES1 as a targetable oncogene in

19 esophageal cancer and describe a new inhibitor for YES1 that has potential for clinical utility

21 Introduction

22 Some cancers depend on a single dominant oncogene for proliferation and survival,

23 a phenomenon called ‘oncogene addiction’ (1). In recent years, some patients with cancers

24 that feature oncogene addiction have greatly benefited from various molecular targeted agents

25 (MTAs). Examples of successful MTAs include anaplastic lymphoma kinase (ALK) inhibitor

1 alectinib for ALK fusion cancer, epidermal growth factor receptor (EGFR) inhibitor erlotinib

2 for EGFR mutant cancer, anti-HER2 antibody trastuzumab for HER2 gene–amplified cancer,

3 and v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib for

4 BRAF mutant cancer (1). These MTAs are dramatically effective in cancers with genetic

5 alterations in the target gene, and their success illustrates why it is important to characterize

6 the cancer type and match the treatment to it. Various MTAs are already approved; however,

7 many cancers with high unmet medical needs still have no clinical MTA options. Esophageal

8 cancer is such a cancer; the 5-year survival rate is less than 20% and clinical MTA options are

9 few, especially for the squamous type (2).

10 SRC, a non-receptor protein tyrosine kinase, has been identified as being encoded by

11 a proto-oncogene, and the nine members of the SRC family, which includes YES1, SRC,

12 FYN, LYN and LCK, have various important cellular functions, such as cell growth, adhesion,

13 survival, and differentiation (3). Reports of genetic alterations in the SRC family are few;

14 however, gene amplification of the site containing YES1 has been reported in clinic (4,5).

15 Furthermore, a recent study found that YES1 gene amplification was a key mechanism of

16 resistance to EGFR or HER2 inhibitors (6,7). Although the transforming ability of YES1 has

17 yet to be proved, there have been various reports of an association between YES1 and

18 tumorigenicity (8). For example, down-regulating YES1 by shRNA significantly inhibited

19 cell growth in several malignancies, including colon carcinoma, rhabdomyosarcoma, and

20 basal-like breast cancer (9-11), and some microRNAs were shown to regulate tumor

21 progression via YES1 regulation (12). Taken together, these reports suggest YES1 may play a

22 key role in cancer progression.

23 Furthermore, there are reports of YES1 participating in numerous signaling

24 pathways. Various mechanisms—including interactions with receptor tyrosine

25 kinases—activate YES1, which in turn activates individual substrates (Focal adhesion kinase

1 (FAK), BCAR1, and paxillin, etc.) resulting in the production of factors involved in growth,

2 survival, invasion, and metastasis (3). In this way, YES1 signaling has been roughly

3 explained, but not fully investigated. Some studies reported that YES1 is associated with

4 YAP1 (13,14). YAP1, originally isolated as a protein that binds to the SH3 domain of c-Yes

5 (15), is known as a transcriptional co-activator in the Hippo pathway and is reported in many

6 studies to be involved in tumor progression (16,17). YAP1 is dysregulated in tumors and its

7 functions include disruption of contact inhibition, tumor progression, suppression of

8 apoptosis, and release from senescence. The activity of YAP1 is downregulated by

9 phosphorylation of five HxRxxS consensus motifs by the large tumor suppressor (LATS)

10 kinase. Serine-phosphorylated YAP1 localizes to cytoplasm and is degraded by proteosomes

11 (16,18). Tyrosine-phosphorylated YAP1 by YES1, SRC, LCK or Abelson murine leukemia

12 viral oncogene homolog (c-ABL) kinases is also reported, but its function seems

13 context-dependent and has not been fully investigated (13,14,19-21).

14 Although selective inhibitors to the SRC family kinases (SFKs) have not yet made

15 their way into clinic, dasatinib and bosutinib are well known as SFK/multikinase inhibitors.

16 These inhibitors also inhibit ABL, which accounts for their approval for the treatment of

17 Philadelphia chromosome-positive chronic myeloid leukemia. Although these inhibitors can

18 kill various types of solid tumor in preclinical models, their off-target toxicity prohibits their

19 use as therapeutics (3,22). Therefore, no SFK inhibitors have so far been approved to treat

20 solid tumors.

21 In this study, we conducteda shRNA library screeningand identified YES1 kinase as

22 an attractive target among the amplified genes in esophageal cancer. We proved the

23 oncogenic activity of YES1, displayed the YES1 gene amplification in clinical, generated an

24 inhibitor that was highly selective against SFK and especially against YES1, and examined

25 its potential efficacy to inhibit YES1 kinase in YES1 gene–amplified tumors. We also

1 evaluated the association of YES1 with YAP1 as a key factor in YES1 gene–amplified cancers

2 and found that YES1 kinase activity regulated YAP1 activity by nuclear translocation and

3 serine phosphorylation, and that YAP1 transcriptional activity contributed to the proliferation

4 of YES1 gene–amplified cancers. In summary, we propose that a YES1-YAP1 blockade by a

5 YES1 inhibitor has therapeutic potential for YES1 gene–amplified cancers.

7 Materials and Methods

8 Cell lines and reagents

9 Cell lines were obtained from the American Type Culture Collection (ATCC),

10 Asterand, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), European

11 Collection of Authenticated Cell Cultures (ECACC), Immuno-Biological Laboratories (IBL),

12 National Cancer Institute (NCI), RIKEN Bio Resource Center (BRC), Health Science

13 Research Resources Bank (HSRRB), Health Protection Agency Culture Collections, Korean

14 Cell Line Bank (KCLB), DS Pharma Biomedical, and the Japanese Collection of Research

15 Bioresources (JCRB) Cell Bank. Esophageal cancer cell lines KYSE70, KYSE140,

16 KYSE-180, KYSE270, KYSE-410, KYSE-450 and KYSE-510 were kindly provided by Dr.

17 Yutaka Shimada (Toyama University) via DSMZ or JCRB (23). All cell lines were obtained

18 more than one year prior to experiments and were propagated for less than 6 months after

19 thawing. All cell lines were cultured according to suppliers’ instructions and confirmed as

20 Mycoplasma negative by culture or PCR methods described elsewhere. Cellular experiments

21 were performed within 20 passages after thawing.

22 Generations of Rat-2/ KYSE70/ OACP4 C/ K562 stable transductants

23 Rat-2 cells (Human science) were infected with an empty lentivirus (Rat-2_mock) or

24 lentivirus containing YES1 wild type (Rat-2_YES1) prepared using

25 pCDH-EF1-MCS-IRES-Puro (System Biosciences). KYSE70, OACP4 C, and K562 were

1 infected with lentiviruses containing YES1 wild type (YES1-WT) or mutant T348I

2 (YES1-GK) prepared using pEZ-Lv105 (GeneCopoeia). Cells stably expressing the above

3 genes were established through tolerance to puromycin following the lentivirus infection.

4 shRNA library screening and data analysis

5 We used Custom Lentiviral shRNA library DECIPHER Human Module 1,

6 DECIPHER Human Module 2, and DECIPHER Human Module 3 (Cellecta). Each module

7 contains ~27,500 shRNAs targeting ~5,000 genes, with 5-6 shRNAs targeting each gene.

8 Further details of the protocol are in Supplementary Materials and Methods. NGS data was

9 analyzed using the Redundant siRNA Activity (RSA) algorithm (24) which calculates

10 gene-centric P values. We defined P <0.05 as a screening hit. We used 4 genes—PLK1,

11 RBX1, KIF11, and EIF3A—as essential genes for positive controls, and luciferase for a

12 negative control.

13 Immunoprecipitation of YES1

14 Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology) supplemented

15 with cOomplete and PhosSTOP (Roche). To immunoprecipitate the YES1 proteins,

16 Dynabeads Protein G (Invitrogen) conjugated to anti-YES1 antibody (Wako, 013-14261) was

17 added to cell lysate and incubated for 2 hours at 4 °C. Precipitates were washed 3 times in the

18 wash buffer, followed by denaturing with the sample buffer solution containing a reducing

19 agent for SDS-PAGE (Life Technologies). The samples were then subjected to SDS-PAGE

20 and analyzed by Western blotting.

21 Western blot analysis

22 Western blot analysis was performed as described previously (25,26). The details of

23 the antibodies are in Supplementary Materials and Methods.

24 Protein kinase assay

25 The inhibitory activity against each kinase was evaluated as described previously

1 (27). The details of the recombinant proteins are in Supplementary Materials and Methods.

2 Cell viability assay

3 Cells were seeded in 96-well plates and incubated at 37 °C with inhibitors. After 4 or

4 7 days, cell viability was measured using Cell Counting Kit-8 solution (Dojindo

5 Laboratories) or CellTiter-Glo Assay (Promega).

6 Mouse xenograft study

7 The mouse xenograft study was performed as described previously (28). All in vivo

8 studies were approved by the Chugai Institutional Animal Care and Use Committee (IACUC).

9 Female BALB/c-nu/nu mice (CAnN.Cg-Foxn1nu/CrlCrlj) were obtained from Charles River

10 Laboratories and kept under specified pathogen-free conditions. Cells (5 × 106 to 7 × 106)

11 were suspended in 200 μL HBSS and injected subcutaneously into the right flank of the mice.

12 For the Rat-2_mock and Rat-2_YES1 models, an equal amount of Matrigel (Corning) was

13 mixed with the cell suspension. Tumor size was measured using a gauge twice per week, and

14 tumor volume (TV) was calculated using the following formula: TV = ab2 /2, where a is the

15 length of the tumor and b is the width. Once the tumors reached a volume of approximately

16 200 to 300 mm3, animals were randomized into groups (n = 3, 4, or 5 in each group), and

17 treatment was initiated. Compound was orally administered once a day.

18 Luciferase reporter assay

19 Cells were co-transfected with TEAD1 luciferase reporter plasmid (pTEAD1-Luc,

20 Signosis) and YAP-irresponsive TK renilla luciferase reporter (pGL4.74-hRluc-TK, Promega)

21 as a reference by using ViaFect (Promega). After 24 hours, cells were treated with the

22 compound for another 24 hours. Cell lysates were analyzed for firefly and renilla luciferase

23 activities with the Dual-Glo assay system (Promega). Firefly luciferase activity was

24 normalized by calculating the ratio to renilla luciferase activity. The relative luciferase

25 activity was reported as a fold change to the compound-untreated control.

1 Real-time quantitative RT-PCR

2 Total RNA was extracted and purified using RNeasy 96 Kit (QIAGEN) and cDNA

3 was synthesized using Transcriptor Universal cDNA Master (Roche) according to the

4 manufacturer’s instructions. Real-time PCR analysis was run on the LightCycler 480 system

5 (Roche). For data analysis, counts were normalized to the housekeeping gene GAPDH at the

6 same condition. Counts are reported as fold change relative to the untreated control. Details

7 of the PCR primers are in Supplementary Materials and Methods.

8 Immunofluorescence

9 Cells were cultured on coverslips or 96-well plates. Cells were fixed, permeabilized,

10 and blocked by Image-iT Fixation/ Permeabilization Kit (Life Technologies). Then cells were

11 incubated with YAP1 antibody (abcam, ab52771) at room temperature for 60 minutes. The

12 secondary antibody was goat anti-Rabbit IgG coupled with Alexa fluor 88 (green) (Life

13 Technologies) incubated at room temperature for 60 minutes in the dark. Nuclei were

14 counterstained with Hoechst 33342. Images were acquired by immunofluorescence

15 microscopy (Nikon, E-600 Eclipse or Thermo Fisher Scientific Inc., CellomicsTM ArrayScan

16 VTI System).

18 Results

19 YES1 has an oncogenic role

20 In a shRNA library of 15,000 genes, we set out to identify an oncogene with a

21 genetic alteration in esophageal cancer, by screening the KYSE70 esophageal cancer cell line,

22 using HCT-116 (colorectal cancer) and NCI-H2009 (lung cancer) cell lines as controls. In the

23 first trial, which compared KYSE70 with HCT-116, the number of genes with a significant

24 impact on growth found only in KYSE70 was 488. In the second trial, which compared

25 KYSE70 with NCI-H2009, the number found only in KYSE70 was 1,164. There were 128

1 genes common to both trials. We screened genetic alterations of the 128 genes in the CCLE

2 database (https://portals.broadinstitute.org/ccle) and identified only one with gene

3 amplification, YES1 (Fig. 1A), which suggested that YES1 could be a target molecule for

4 esophageal cancer therapy.

5 According to an in vitro experiment in a previous publication, YES1 had no

6 oncogenic activity (8). However, we proved YES1’s oncogenic activity with in vivo

7 experiments. We lacked access to esophageal cancer cell lines with YES1 deletion, so we used

8 the normal rat fibroblast cell line Rat-2 to generate clones that had been stably transduced

9 with an empty lentiviral vector (Rat-2_mock) or a lentiviral vector expressing YES1

10 (Rat-2_YES1). The use of a normal fibroblast cell line to validate oncogenic activity is a

11 common procedure for validating transformation of oncogenes such as ALK fusion, ROS

12 fusion and FGFR fusion(28-30). We used the YES1-transduced bulk cells and checked YES1

13 expression in Rat-2_YES1 cells in vitro and in vivo (Figs. 1B-C). We observed that YES1

14 over-expression promoted tumorigenicity in Rat-2_YES1 cells compared to the Rat-2_mock

15 cells (Fig. 1D), and Rat-2_YES1 tumors were about 13 times larger than Rat-2_mock tumors

16 at 45 days after inoculation (Fig. 1E), suggesting that YES1 has oncogenic potential in vivo.

18 YES1 gene amplification occurs in various cancer types

19 To evaluate the frequency of YES1 genetic alterations in clinical, we analyzed copy

20 number (CN) alterations and mutations in TCGA data using cBioPortal

21 (http://www.cbioportal.org/) (31,32) and found YES1 gene amplification in various tumor

22 types (Table 1), but since the biological function of each YES1 mutation is unknown, we did

23 not analyze them in detail. We confirmed YES1 gene amplification by FISH in a tissue

24 microarray of esophageal cancer and lung cancer (Supplementary Fig. S1A). Next, we used

25 the PrognoScan database (33) to analyze the correlation between YES1 expression and

1 prognosis for these tumor types. In esophageal and lung cancer, the prognosis for patients

2 with high YES1 expression was worse than for those with low YES1 expression at a level

3 that is statistically significant, suggesting the contribution of YES1 in these malignancies

4 (Supplementary Fig. S1B). Taken together, we hypothesized that YES1 gene amplification

5 accompanied with high expression could be an anticancer target, not only for esophageal

6 cancer but also for other cancers, and we set about generating a YES1 inhibitor.

8 YES1 kinase inhibitory activity of CH6953755

9 To obtain a YES1 inhibitor, we performed a high-throughput screening of a chemical

10 library with more than half-a-million compounds at Chugai Pharmaceutical Co. Ltd. and

11 identified a lead compound that inhibited a relatively broad range of kinases but had a

12 different chemical scaffold from known SFK inhibitors. We then structurally modified and

13 improved the pharmacokinetics profile and selectivity against SFKs, especially YES1. Finally,

14 we generated an aminopyrazole derivative, CH6953755, as a potent and orally available

15 YES1 inhibitor (34) (Fig. 2A).

16 To investigate the selectivity of CH6953755 and compare it with other known SFK

17 inhibitors, dasatinib (35) and bosutinib (36), we evaluated the inhibitory activity against 39

18 kinases in an enzyme assay (Figs. 2A-B and Supplementary Table. S1). The IC50 value of

19 CH6953755 on the enzyme activity of YES1 was 1.8 nmol/L. Although bosutinib had similar

20 IC50 value as CH6953755 and dasatinib had about 10-fold higher activity on YES1 kinase

21 inhibition, CH6953755 exhibited more specificity for YES1 among the tested SFKs

22 compared to both compounds (Figs. 2B-C and Supplementary Table. S1). We used dasatinib

23 for a further comparison study of CH6953755 based on its potent YES1-inhibiting activity

24 but low selectivity.

25 To evaluate the kinase selectivity of CH6953755 further, we used a KINOMEscan

1 panel (DiscoveRx) consisting of 456 WT and mutant kinases (Supplementary Table S2). In

2 the panel, CH6953755 at 10 nmol/L bound (at more than 65% inhibition to an ATP analog) to

3 only 8 WT kinases, including YES1 and 4 other SFKs.

5 YES1 kinase inhibition leads to cell growth inhibition of YES1-amplified cancer cell

6 lines

7 Next, we investigated whether YES1 kinase inhibition inhibited the cell growth of

8 YES1-amplified cancer cells. First, we analyzed the YES1 expression and CN data of 1035

9 cancer cell lines in the CCLE database to determine the profile of lines harboring YES1 gene

10 amplification. We defined YES1 gene amplification as having a copy number over four. We

11 confirmed that YES1 expression was upregulated in amplified cell lines (Supplementary Fig.

12 S2A), and validated this upregulation by measuring YES1 protein levels in four

13 YES1-amplified and four non-YES1-amplified cancer cell lines (Supplementary Fig. S2B).

14 These results confirmed that YES1 gene amplification includes the upregulation of YES1

15 expression in YES1-amplified cancer cell lines both in mRNA and protein levels. Next, we

16 assessed the anti-proliferative activity of YES1 inhibitors. CH6953755, dasatinib, EGFR

17 inhibitor erlotinib, mTOR inhibitor everolimus, and a MEK inhibitor CH4987655 (37) were

18 tested against a panel of 66 cancer cell lines, seven of which had YES1 amplification.

19 YES1-amplified cancer cell lines showed higher sensitivity to CH6953755 than non–

20 YES1-amplified cancer cell lines (Fig. 3A and Supplementary Table S3). CH6953755

21 prevented the autophosphorylation at Tyr426 of YES1 that upregulates enzymatic activity in

22 KYSE70 cells harboring YES1 amplification (Fig. 3B). Dasatinib also showed more potent

23 efficacy against YES1-amplified cell lines than against non-amplified lines (Supplementary

24 Fig. S3A), but YES1 amplification did not seem to predict the efficacy of erlotinib,

25 everolimus, or CH4987655 (Supplementary Figs. S3B-D, Supplementary Table S2).

1 Therefore, we considered that YES1 gene amplification status could predict the efficacy of a

2 YES1 inhibitor. Next, we tried to check whether the CH6953755 cytotoxicity was the result

3 of YES1 inhibition in YES1-amplified cancer cell lines using the commonly used

4 chemical-genetic approach of engineering an inhibitor-resistant mutant of YES1 (38,39).

5 From YES1-amplified KYSE70 and OACP4 C, and YES1 non-amplified K562 cell lines, we

6 generated clones stably transduced with a lentiviral vector expressing YES1 wild type

7 (YES1-WT) or mutant T348I, a so-called gatekeeper mutation (YES1-GK) that had a point

8 mutation at the binding site with CH6953755 and was expected to be resistant to CH6953755.

9 In KYSE70 cells expressing YES1-GK (KYSE70_YES1-GK), phosphorylation at Tyr426

10 was not suppressed by CH6953755 treatment, unlike in KYSE70 cells expressing YES1-WT

11 (KYSE70_YES1-WT) (Fig. 3C). As we expected, the two cell lines expressing YES1-GK

12 with amplified YES1 gene became resistant to CH6953755, while this was not the case with

13 the line without amplified YES1 (Fig. 3D). Similar results were found with dasatinib

14 (Supplementary Fig S4). These results indicated that YES1 kinase inhibition inhibited the

15 cell growth of YES1-amplified cancer cell lines.

16 Furthermore, we analyzed whether the other SFKs had an effect on YES1-amplified

17 cancer cell lines. We examined SFK expression profiles in YES1-amplified cancer cell lines

18 and determined the dominance of YES1 expression (Supplementary Fig. S5), suggesting that

19 the contributions of SFKs other than YES1 were low in YES1-amplified cancer cell lines. We

20 focused on SRC as representative of other SFKs and confirmed this with a cell growth

21 inhibition assay using KYSE70 clones stably transduced with an empty lentivirus or

22 lentivirus expressing SRC-WT or YES1-WT (KYSE70_mock, KYSE70_SRC-WT,

23 KYSE70_YES1-WT) treated with CH6953755 or dasatinib. Efficacy against KYSE70_mock

24 and KYSE_YES1-WT was almost the same for treatment with CH6953755 and dasatinib, but

25 the efficacy of CH6953755 in KYSE70_SRC-WT was less sensitive than in

1 KYSE70_YES1-WT or KYSE70_mock, while the efficacy of dasatinib was pretty much

2 unchanged (Supplementary Table S4). It was thought that SRC initiated activation of the

3 same downstream signal as YES1 which contributed to cell proliferation when SRC was

4 expressed in KYSE70, and SRC inhibitory activity of CH6953755 was about 20-times

5 weaker than YES1 inhibitory activity, while the SRC inhibitory activity of dasatinib was

6 almost same as YES1 (Supplementary Table S1). Based on the evidence from these

7 experiments, we think that YES1 activity is dominant among SFKs, and that the cytotoxicity

8 of CH6953755 resulted from YES1 inhibition in YES1-amplified cancer cell lines, all of

9 which suggests the therapeutic potential of a YES1 inhibitor for YES1-amplified cancers.

11 YES1 kinase inhibition suppresses the growth of YES1-amplified xenograft tumors

12 To confirm whether YES1 inhibition led to antitumor activity in vivo as well as cell

13 growth inhibition in vitro, first we evaluated the in vivo efficacy of YES1 inhibitor

14 CH6953755 in a Rat-2_YES1 xenograft mouse model. After oral treatment of CH6953755 at

15 60 mg/kg, we observed antitumor activity (Fig. 4A) accompanied with phospho-Tyr426

16 YES1 suppression in xenograft tumors (Fig. 4B). Next, we utilized native cancer cell lines

17 harboring YES1 gene amplification. Consistent with the findings in the Rat-2_YES1 model,

18 CH6953755 showed antitumor activity against xenograft models with YES1 amplification,

19 such as KYSE70 (CN=9.9, maximum tumor growth inhibition (TGI) 90% at the maximum

20 tolerated dose (MTD)) and RERF-LC-AI (CN=5.1, maximum TGI 93% at MTD) (Fig. 4C).

21 In contrast, CH6953755 did not show antitumor activity against xenograft models with no

22 YES1 amplification, such as ACHN (CN=1.8, maximum TGI 13% at MTD) and HARA

23 (CN=2.7, maximum TGI 13% at MTD) (Fig. 4D). These data were in line with the in vitro

24 observations. We also tested dasatinib against a YES1-amplified KYSE70 model. Because its

25 toxicity limits the dose, dasatinib showed moderate efficacy (maximum TGI 41% at MTD)

1 (Supplementary Fig S6). Then we evaluated the suppression of YES1 signaling in tumor

2 tissues by conducting Western blotting 24 hours after the last administration of daily dosing.

3 The blots demonstrated that CH6953755 suppressed phospho-Tyr426 YES1 in a

4 dose-dependent manner (Fig. 4E). These results suggest that YES1 kinase inhibition by

5 CH6953755 leads to selective antitumor activity against YES1-amplified cancers both in vitro

6 and in vivo, and YES1 kinase plays a crucial role in YES1-amplified cancers.

8 YAP1 plays a role downstream of YES1 in YES1-amplified cancer

9 Next, we investigated how YES1 inhibition achieved antitumor activity, focusing on

10 YAP1. Since genetic alterations in the hippo pathway, such as LATS2 gene deletion, result in

11 YAP1 activation that leads to tumor progression (40), we hypothesized that YES1 gene

12 amplification is a non-canonical YAP1 activation mechanism in cancer cells. The

13 transcription factor TEAD has been reported to bind YAP1 and play a major role in YAP1

14 oncogenic function (41). YAP1 functions as a transcriptional coactivator for TEAD to induce

15 gene expression, such as ANKRD1 and CYR61, thereby promoting cell growth, proliferation,

16 and survival (16,42). Therefore, we transfected the cell lines with TEAD luciferase reporter

17 and tested the effect of YES1 inhibitor CH6953755 on luciferase activity. CH6953755

18 suppressed TEAD luciferase reporter activity in YES1-amplified KYSE70 and RERF-LC-AI

19 (Fig. 5A). To confirm that the suppression was derived from YES1 kinase inhibition, we

20 utilized KYSE70_YES1-GK and found that CH6953755 could not suppress their TEAD

21 luciferase reporter activity, even though it could suppress the activity in KYSE70_YES1-WT

22 (Fig. 5B). Furthermore, CH6953755 suppressed the expression of YAP1 downstream genes,

23 ANKRD1 and CYR61, in KYSE70_ YES1-WT, without affecting the YAP1 expression level,

24 and this suppression was abrogated in KYSE70_ YES1-GK (Fig. 5C). These phenomena

25 were also observed with dasatinib treatment (Supplementary Figs. S7A-B), suggesting that

1 YES1 kinase activity regulates YAP1 activity.

3 YES1 regulates YAP1 activity by controlling its nuclear translocation and serine

4 phosphorylation via kinase activity

5 Our next question was how YES1 regulated YAP1 activity. First we looked at the

6 YAP1 localization. Multikinase inhibitor dasatinib has been reported to inhibit nuclear

7 localization of YAP1 (20,21), so we hypothesized that YES1 regulated YAP1 activity by

8 controlling YAP1 localization. We evaluated the localization of YAP1 and confirmed the

9 phenomenon in KYSE70_YES1-WT or -GK after treatment with CH6953755. YAP1 nuclear

10 localization was inhibited in the KYSE70 cell line expressing YES1-WT, and the inhibition

11 was abrogated in KYSE70_ YES1-GK (Fig. 6A and Supplementary Fig. S8). Furthermore,

12 we confirmed this phenomenon in in vivo setting. (Supplementary Fig. S9). Next, we

13 evaluated the effects of YAP1 phosphorylation sites on CH6953755 activity. Prior work has

14 demonstrated that YES1 phosphorylation of YAP1 at the tyrosine 357 site regulated YAP1

15 transcriptional activity (13), so we examined the TEAD luciferase reporter activity when the

16 following YAP1 mutations were being expressed: Y357E, which is phosphomimetic, or

17 Y357F, which is phosphodead (19). Neither the YAP1 Y357E nor the Y357F mutation

18 affected the suppression of YAP1 transcriptional activity by CH6953755 (Fig. 6B), which

19 suggested that phosphorylation of YAP1 at tyrosine 357 did not have any impact on the

20 control of YAP1 activity by YES1 in YES1-amplified cancer. Then we assessed the effect of

21 YAP1 serine sites, which are important for YAP1 regulation in the canonical Hippo pathway.

22 Several groups have reported that YAP1 5SA, which is mutated in all of the HxRxxS

23 consensus motifs, makes YAP1 constitutively active by abolishing the phosphorylation by

24 LATS (43), so we generated two KYSE70 clones, #1 and #7, which were stably transduced

25 with a lentiviral vector expressing YAP1 5SA. In these clones, the suppression of TEAD

1 luciferase reporter activity after treatment with CH6953755 was abrogated (Fig. 6C), and the

2 antiproliferative activity of CH6953755 was diminished (Fig. 6D). Taken together, the

3 findings suggest that YES1 regulates YAP1 activity by serine phosphorylation indirectly and

4 by nuclear localization. In conclusion, YES1-YAP1 signal inhibition by CH6953755 leads to

5 antitumor activity in YES1-amplified cancer cells.

7 Discussion

8 In this study, first we screened an shRNA library to identify YES1 as a fascinating

9 oncogenic target, and we also analyzed the Project Achilles data (44). The frequency of YES1

10 gene amplification among cell lines in Project Achilles is 2.6% (13/502). We used four genes,

11 PLK1, RBX1, KIF11 and EIF3A, as essential genes for positive control, but these genes

12 showed no significance in YES1-amplified cancer cell lines based on our analysis of Achilles

13 data. Next, we changed the criteria for positive control by using the average of the most

14 potent three of the four individual EIF3A shRNAs to show significance (P<0.05). Four out of

15 13 YES1-amplified cancer cell lines met this criterion, but we did not identify either YES1

16 nor YAP1 as hits. This result differs from our shRNA screening result, but we think that this

17 is due to the different experimental and analytical conditions, as well as the possibility of

18 lower silencing efficiency of YES1 shRNAs and YES1 CN in the analyzed cell lines with a

19 lower CN than KYSE70. We determined that we could confirm our shRNA screening result

20 showing that YES1 had an important role in KYSE70 by using the chemical-genetic approach

21 of engineering an inhibitor-resistant GK mutant of YES1 (Figure 3D).

22 We demonstrated that YES1 has oncogenic potential and is activated by gene

23 amplification in several tumor types. Therefore, patients with the amplified YES1 gene are

24 expected to benefit from a YES1 inhibitor. At present, the well-known SFK/multikinase

25 inhibitors dasatinib and bosutinib are not effective against solid tumors in clinical. One

1 often-cited reason is that there are no prognostic biomarkers related to SFK activity that can

2 be used to select patients for clinical trials (3,22). However, even if an appropriate biomarker

3 was found, the regimens needed with these inhibitors when treating chronic myeloid

4 leukemia are already close to the MTD but would still be insufficient to inhibit

5 phosphorylation of SRC (45,46). Consistent with the finding in clinical, dasatinib even at the

6 MTD achieved moderate in vivo efficacy against YES1-amplified KYSE70 xenograft model

7 (Supplementary Fig. S6), whereas treatment with CH6953755 achieves significant efficacy at

8 a non-toxic dose (Fig. 4C). These data suggest that the selectivity of CH6953755 will allow

9 the dose in clinic to be increased to a level sufficient to suppress YES1 kinase and that

10 CH6953755 could be effective in meeting the high unmet medical needs of patients with

11 YES1-amplified cancer.

12 The YES1 gene might also be enriched in patients insensitive to several current

13 standards of care. One study reported that treatment with 5-fluorouracil (5-FU) upregulated

14 YES1 and YAP1 in colorectal cancer patients and these patients have a poor prognosis (47).

15 Treatment with 5-FU was also reported to increase the gene CN of the region that includes

16 thymidylate synthetase (TYMS), a target of 5-FU, in colorectal cancer patients, and this

17 increase is considered to be one of the mechanisms of 5-FU resistance (48). As the

18 chromosome locus of TYMS is so close to YES1, the YES1 gene could be amplified together

19 with the TYMS gene. Since 5-FU is the first-line therapy in esophageal cancer, this pattern of

20 resistance could also happen there. Although esophageal cancer patients in general have quite

21 a poor prognosis, the segment of patients with high YES1 expression have a worse prognosis

22 than the segment with low expression (Supplementary Fig. S1B). Furthermore, YES1

23 amplification is a mechanism of resistance to some MTAs (6,7). For example, acquired

24 amplification of YES1 is a recurrent mechanism of resistance to EGFR inhibition in

25 EGFR-mutant lung cancers for which YES1 inhibition is effective (49). Moreover, reports

1 show that YAP1 is essential for the suppression of anti-tumor immunity (50). These lines of

2 evidence suggests the presence of YES1 gene amplification in groups that are insensitive to

3 chemotherapy and MTAs, also suggesting that YES1-amplified cancers are not sensitive to

4 immune checkpoint inhibitors. Therefore, we think that CH6953755 will provide benefit to

5 these segments.

6 We revealed that YES1 regulates YAP1 activity by controlling YAP1 localization,

7 and that the YES1-YAP1 signal is important in the proliferation of YES1-amplified cancers.

8 In the cell growth inhibition assay, the YES1-amplified cell line KYSE-510 was insensitive to

9 CH6953755 and dasatinib (Supplementary Table S3), and harbored not only YES1 gene

10 amplification but also YAP1 gene amplification. This suggests that YES1 could exist

11 upstream of YAP1, which also supports our findings. However, two points are still unclear:

12 How precisely does YES1 regulate YAP1 activity? Are there other important YES1

13 downstream signals in YES1-amplified cancers? Regarding YAP1 regulation by YES1,

14 previous research found that YES1 directly phosphorylated YAP1 at tyrosine 357 and

15 regulated YAP1 transcriptional activity in β-catenin-driven cancers (13). In contrast to

16 previous research, our evaluation of YES1-amplified cancer expressing phosphomimetic

17 Y357E and phosphodead Y357F showed that tyrosine 357 phosphorylation did not contribute

18 to YAP1 transcriptional activity (Fig. 6B). On the other hand, our evaluation in

19 YES1-amplified cancer expressing YES1-GK proved that YES1 kinase activity is

20 indispensable for YAP1 transcriptional activity and nuclear translocation (Figs. 5B-C and Fig.

21 6A), and the evaluation in YES1-amplifed cancer expressing five serine-mutated YAP1

22 implies that YAP1 serine phosphorylation has an effect on the YES1-YAP1 signal (Figs.

23 6C-D). These results suggest that YES1 may indirectly activate YAP1 via phosphorylation of

24 some other kinase involved in the regulation of YAP1 serine phosphorylation, which in turn

25 suggests that the mechanism by which YES1 regulates YAP1 may be dependent on the

1 cellular context. As for other YES1 downstream signals important for proliferation, we

2 consider that signals other than YAP1 do contribute to the proliferation of YES1-amplified

3 cancers. In cells expressing the YAP1 constitutive active form, YAP1 5SA, suppression of

4 YAP1 transcriptional activity by CH6953755 was abrogated completely, but the abrogation of

5 cell growth inhibition was partial (Figs. 6C-D). Therefore, we think that known YES1

6 downstream signals, such as MAPK and AKT, or other signals may be involved in the

7 proliferation of YES1-amplified cancers.

8 In summary, we suggest that YES1-YAP1 signal inhibition is a mode of action

9 through which CH6953755 achieves efficacy, and that CH6953755 has therapeutic potential

10 for patients harboring the YES1 gene amplification.

12 Acknowledgments

13 We thank Yasue Nagata, Maiko Izawa, Yumiko Hashimoto, Tomoaki Hayashi,

14 Yusuke Ide, Fumie Sawamura for performing pharmacological assays, Chiaki Senoo, Hiroshi

15 Tanaka, Kenji Kashima, Hironori Mutoh, Yuko Aoki, Masahiro Aoki and Takuo Tsukuda for

16 helpful discussion, and Hideaki Mizuno for PrognoScan analysis.

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2 Table 1. The clinical prevalence of YES1 gene amplification in various cancer types. 3 Cancer type Frequency

Esophageal Squamous Cell Carcinoma (TCGA, Provisional) 6.3% (6/96 cases)

Esophageal Adenocarcinoma (TCGA, Provisional) 5.7% (5/88 cases)

Head and Neck Squamous Cell Carcinoma (TCGA, Provisional) 5.1% (27/530 cases)

Lung Squamous Cell Carcinoma (TCGA, Provisional) 4.5% (23/511 cases)

Bladder Urothelial Carcinoma (TCGA, Provisional) 4.4% (18/413 cases)

Sarcoma (TCGA, Provisional) 3.0% (8/265 cases)

Ovarian Serous Cystadenocarcinoma (TCGA, Provisional) 3.0% (18/606 cases) 4 5

2 Figure legends

4 Table 1. The clinical prevalence of YES1 gene amplification in various cancer types. We

5 analyzed the clinical copy number alteration data using cBioPortal

6 (http://www.cbioportal.org/) on August 18th, 2018. The results shown here are based upon

7 data generated by the TCGA Research Network (https://cancergenome.nih.gov/).

9 Figure 1. The shRNA library screening identified YES1 as an oncogene for an esophageal

10 cancer cell line. A. Venn diagrams show the number of genes with a significant impact on

11 growth (P value <0.05). The criterion for amplification is an amplification ratio of >2.0. B.

12 Rat-2 parent, Rat-2_mock, and Rat-2_YES1 cells were lysed and analyzed by Western blot.

13 YES1 proteins were immunoprecipitated from cell lysates and blotted. C. Xenograft tumors

14 derived from implantation of Rat-2_mock and Rat-2_YES1 cells into mice were lysed and

15 analyzed by Western blot (n=3, 4, respectively). YES1 proteins were immunoprecipitated

16 from cell lysates and blotted. D. Rat-2_mock and Rat-2_YES1 cells (5 × 106) were implanted

17 into nude mice. Tumor volumes in each group were measured. Data are shown as mean ± SD

18 (n=10, 7). Two-tailed Student’s t-test: ***p < 0.001, versus Rat-2_mock at final day. E.

19 Representative images of Rat-2_mock and Rat-2_YES1 mice. Tumor volumes in each group

20 were measured at day 45 after inoculation (n=10, 7).

22 Figure 2. Compared to dasatinib and bosutinib, CH6953755 tends to be selective to YES1. A.

23 Chemical structures of CH6953755, dasatinib, and bosutinib. B. Kinase inhibitory activity of

24 CH6953755, dasatinib, and bosutinib against 39 kinases. C. Kinase inhibitory activity of

25 CH6953755, dasatinib, and bosutinib against 5 SFKs.

2 Figure 3. YES1 kinase inhibition inhibits the cell growth of YES1-amplified cancer cell lines.

3 A. The IC50 value of CH6953755 was evaluated in a cell proliferation assay. In total, 7

4 YES1-amplified and 59 non–YES1-amplified cancer cell lines were analyzed. Two-tailed

5 Student’s t-test: *** p<0.001. B and C. The indicated concentrations of CH6953755 were

6 applied for 2 hours to (B) the KYSE70 cell line, and (C) cloned cell lines,

7 KYSE70_YES1-WT and KYSE70_YES1-GK. The cells were lysed and analyzed by Western

8 blot. YES1 proteins were immunoprecipitated from cell lysates and blotted. D.

9 YES1-amplified cancer cell lines KYSE70 and OACP4 C, and non–YES1-amplified cancer

10 cell line K562 expressing YES1-WT or YES1-GK were seeded in 96-well plates and treated

11 for 4 days with various concentrations of CH6953755. Subsequently, cell viability was

12 measured.

14 Figure 4. CH6953755 shows selective antitumor activity against YES1-amplified xenograft

15 tumors. A and B. Mice bearing Rat-2_YES1 cells were treated with CH6953755 60 mg/kg or

16 vehicle orally once daily for 10 days. (A) Tumor volume in each group was measured. Data

17 are shown as mean ± SD (n=3). Two-tailed Student’s t-test: *p < 0.05, versus vehicle

18 treatment at final day. (B) Xenograft tumors were extracted 24 hours after the last dosing and

19 analyzed by Western blotting (n=3). YES1 proteins were immunoprecipitated from cell

20 lysates and blotted. C, D, and E. CH6953755 at the indicated doses was given orally once

21 daily to (C) xenograft models bearing cells with YES1 amplification, KYSE70 (for 11 days)

22 and RERF-LC-AI (for 14 days) or (D) xenograft models bearing cells without YES1

23 amplification, ACHN (for 12 days) and HARA (for 11 days). Tumor volume and body weight

24 change in each group were measured. Data are shown as mean ± SD (n=4). (C) Parametric

25 Dunnett’s test: ***p < 0.001, **p < 0.01, *p < 0.05, n.s., no significant difference, versus

1 vehicle treatment at final day. (D) Two-tailed Student’s t-test: n.s., no significant difference,

2 versus vehicle treatment at final day. (E) Xenograft tumors of KYSE70 (n=4, 3) and

3 RERF-LC-AI (n=4, 2) were extracted 24 hours after the last dosing and analyzed by Western

4 blotting. YES1 proteins were immunoprecipitated from cell lysates and blotted.

6 Figure 5. YAP1 plays a role downstream of YES1 in YES1-amplified cancer. A and B. The

7 effect of a YES1 inhibitor on luciferase activity was tested in (A) KYSE70, RERF-C-AI, and

8 (B) KYSE70_YES1-WT and KYSE70_YES1-GK cells. Cells were co-transfected with

9 TEAD1-luciferase and renilla luciferase reporters. After 24 hours, cells were treated with the

10 indicated concentrations of CH6953755 for 24 hours, and the relative luciferase activity was

11 determined. Data are shown as mean ± SD (n=3). Parametric Dunnett’s test: ***p < 0.001,

12 **p < 0.01, *p < 0.05, n.s., no significant difference, versus DMSO control treatment for each

13 cell line. C. KYSE70_YES1-WT and KYSE70_YES1-GK were treated with the indicated

14 concentrations of CH6953755 for 24 hours. Gene expression levels were determined by

15 quantitative PCR. Data are shown as mean ± SD (n=3). Parametric Dunnett’s test: ***p <

16 0.001, **p < 0.01, n.s., no significant difference, versus DMSO control treatment for each

17 cell line.

19 Figure 6. YES1 regulates YAP1 activity by controlling its nuclear translocation and serine

20 phosphorylation. A. KYSE70_YES1-WT and KYSE70_YES1-GK cells were treated with

21 CH6953755 1 μM for 24 hours. The subcellular location of YAP1 was determined by

22 immunofluorescence. High magnification of the area marked within the frame is in

23 Supplementary Fig. S8. B. KYSE70 cells were co-transfected with TEAD1-luciferase and

24 renilla luciferase reporters with YAP1_WT, YAP1_Y357F, or YAP1_Y357E plasmids. After

25 24 hours, cells were treated with the indicated concentrations of CH6953755 for 24 hours,

1 and the relative luciferase activity was determined. Data are shown as mean ± SD (n=3).

2 Parametric Dunnett’s test: *p < 0.05, n.s., no significant difference, versus YAP1_WT for

3 each compound concentration. C. KYSE70, KYSE70_YAP1-5SA #1, and

4 KYSE70_YAP1-5SA #7 cells were co-transfected with TEAD1-luciferase and renilla

5 luciferase reporters. After 24 hours, cells were treated with the indicated concentrations of

6 CH6953755 for 24 hours, and the relative luciferase activity was determined. Data are shown

7 as mean ± SD (n=3). Parametric Dunnett’s test: ***p < 0.001, n.s., no significant difference,

8 versus DMSO control treatment for each cell line. D. KYSE70, KYSE70_YAP1-5SA #1 and

9 KYSE70_YAP1-5SA #7 cells were seeded in 96-well plates and treated for 4 days with

10 various concentrations of CH6953755. Subsequently, cell viability was measured. Data are

11 shown as mean ± SD (n=3). Parametric Dunnett’s test: ***p < 0.001, versus KYSE70 cell at

12 final day.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Figure 1 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A HCT-116 KYSE70 KYSE70 NCI-H2009

520 243 488 1164 219 625

KYSE70 KYSE70 360 128 1036

Only one gene hit with amplification: YES1

B C Mock YES1 Parent Mock YES1 IP: YES1 IP: YES1 IB: pY416 SFK IB: pY416 SFK

IB: YES1 IB: YES1

input input IB: GAPDH IB: GAPDH

D E 2400 Rat-2_MockRat-2_mock Rat-2_mock Rat-2_YES1 Rat-2_YES1Rat-2_YES1 2000 ) 3

1600

1200

800

Tumor volume volume Tumor (mm 400 *** 0 32 34 36 38 40 42 44 46 Average Average 3 3 Day after the inoculation 159 ± 124 mm 1574 ± 619 mm (n =10) (n=7)

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Figure 2 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A CH6953755 Dasatinib Bosutinib

B CH6953755 IC50 (mmol/L) Dasatinib IC50 (mmol/L) Bosutinib IC50 (mmol/L) 0.001 0.1 10 0.00001 0.001 0.1 10 0.001 0.1 10 YES1 YES1 YES1 LCK LCK LCK FYN FYN FYN LYN LYN LYN SRC SRC SRC EGFR EGFR EGFR ABL ABL ABL TRKC TRKC TRKC EPHB2 EPHB2 EPHB2 EPHB4 EPHB4 EPHB4 HER2 HER2 HER2 PDGFR PDGFR PDGFR KIT KIT KIT BRAF BRAF BRAF CRAF CRAF CRAF EphA2 EphA2 EphA2 p38 alpha p38 alpha p38 alpha FGFR2 FGFR2 FGFR2 JAK2 JAK2 JAK2 KDR KDR KDR FLT3 FLT3 FLT3 IRAK1 IRAK1 IRAK1 ALK ALK ALK AXL AXL AXL CHK2 CHK2 CHK2 FAK FAK FAK MEK1 MEK1 MEK1 CHK1 CHK1 CHK1 PKC alpha PKC alpha PKC alpha PKC beta1 PKC beta1 PKC beta1 CDK1 CDK1 CDK1 MET MET MET INSR INSR INSR AKT1 AKT1 AKT1 PKCi PKCi PKCi AuroraA AuroraA AuroraA MNK1 MNK1 MNK1 ERK1 ERK1 ERK1 PKA PKA PKA

C CH6953755 Dasatinib Bosutinib 20 20 20

18 18 18

50 16 50 16 50 16 14 14 14 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Ratio to YES1IC Ratio 2 to YES1IC Ratio 2 to YES1IC Ratio 2 0 0 0

Figure 3

A B C C H 6 9 5***3 7 5 5 3 D

1 0 0 KYSE70_ KYSE70_ YES1-WT YES1-GK

]

1 0

/L) M

CH6953755 CH6953755 m

[

(mmol/L) (mmol/L) IP: YES1 0 0.001 0.003 0.01 0.03 0.1 0.3 1 0 0.001 0.02 0.3 5 0 0.001 0.02 0.3 5

IP: YES1 e

mol u m l 1

( a

v IB: pY416 SFK IB: pY416 SFK

0 5

IC C

I 0 . 1 IB: YES1 IB: YES1

input input IB: GAPDH IB: GAPDH 0 . 0 1

p p m m YES1 a amp YES1 non-a -amp S n E o Y n S 120 E Y D KYSE70 OACP4 C 100 K562 120 (YES1 amp) (YES1 amp) 120 (YES1 non-amp) 120 80

100 100 100 60 (%) 80 80 YES1-WT 80 40 60 60 YES1-GK 60 20 viability (%) viability 40 40 40 0 Cell viability (%) viability Cell Cell viability Cell 0.0001 0.1 20 20 20

0 0 0 0.0001 0.001 0.01 0.1 1 0.0001 0.001 0.01 0.1 1 0.0001 0.001 0.01 0.1 1 Concentration (mmol/L) Concentration (mmol/L) Concentration (mmol/L)

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Figure 4 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B 1600 Vehicle CH6953755

) 1400 3 60 mg/kg Vehicle 60 mg/kg 1200 IP: YES1 1000 IB: pY416 SFK 800 600 IB: YES1

400 input Tumor volume volume Tumor (mm 200 * IB: YES1 0 29 31 33 35 37 39 IB: Vinculin Day after the inoculation

C D KYSE70 RERF-LC-AI ACHN HARA (YES CN=9.9) (YES CN=5.1) (YES CN=1.8) (YES CN=2.7) 900 1400 450 900

800 400 800 ) 1200 ) 3 700 3 350 700 1000 600 300 n.s. 600 n.s. 500 800 250 500 * n.s. 400 600 200 400 300 *** * 150 300 *** 400 200 *** ** 100 200

Tumor volume (mm volume Tumor ** (mm volume Tumor 100 200 50 100 0 0 0 0 9 11 13 15 17 19 21 23 11 13 15 17 19 21 23 19 22 25 28 31 34 14 16 18 20 22 24 26

120 120 120 120

100 100 100 100

80 80 80 80 Body weight (%) weight Body 11 13 15 17 19 21 23 19 22 25 28 31 34 (%) weight Body 9 11 13 15 17 19 21 23 14 16 18 20 22 24 26 Day after the inoculation Day after the inoculation Day after the inoculation Day after the inoculation

vehicle 7.5 mg/kg Vehicle Vehicle 60 mg/kg 15 mg/kg 30 mg/kg 60 mg/kg 450 400 1400 CH6953755 CH6953755 E (mg/kg) 350 (mg/kg) 1200 Vehicle 7.5 15 30 60 Vehicle300 7.5 15 30 60 1000 250 YES1 IP: IB: 800pY416 SFK 200

IB: YES1 600 150 input IB: GAPDH 400 100

200 KYSE70 50 RERF-LC-AI 0 0 19 21 23 25 27 29 31 33 9 14 19

Figure 5

A B KYSE70_YES1-WT KYSE70_YES1-GK KYSE70 RERF-LC-AI 1.6

1.2 1.2 n.s.

1.4 * 1 n.s. 1 1.6 1.2 1.4 n.s. n.s. 1.21 n.s. 0.60.8 0.8 0.8 1 0.4 0.20 0.8 0 1 0.6 0.6 * 0.1 0.01 0.001 *** ** 0.6 0.4 *** 0.4 CH6953755 *** *** Relative mRNA 0.4 *** *** *** *** Relative luciferaseactivity 0.2 Relative luciferaseactivity 0.2 Relative luciferase activity 0.2

0 0 0 0 0.1 0.3 1 3 0 0.1 0.3 1 3 0 0.1 0.3 1 3 CH6953755 (mmol/L) CH6953755 (mmol/L) CH6953755 (mmol/L) C 1.6 YAP1 1.6 CYR61 1.6 ANKRD1 n.s. 1.4 1.4 1.4 n.s. n.s. n.s.

1.2 n.s. n.s. 1.2 n.s. 1.2 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 1 n.s. 1 1 n.s. ** *** n.s. 0.8 0.8 0.8 *** 0.6 0.6 0.6 Relative mRNA 0.4 Relative mRNA 0.4 Relative mRNA 0.4 *** *** *** 0.2 0.2 0.2

0 0 0 0 0.001 0.01 0.1 1 0 0.001 0.01 0.1 1 0 0.001 0.01 0.1 1 CH6953755CH6953755 (mmol/L) CH6953755CH6953755 (mmol/L) CH6953755CH6953755 (mmol/L)

KYSE70_YES1-WT KYSE70_YES1-WT KYSE70_YES1-WT KYSE70_YES1-GK KYSE70_YES1-GK KYSE70_YES1-GK Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 6 KYSE70_ KYSE70_ A YES1-WT YES1-GK B 1.2 YAP1_WT

1 n.s. YAP1_Y357F n.s. DMSO YAP1_Y357E * 0.8 n.s. n.s. 50mm 50mm 0.6 n.s. n.s. n.s. 0.4 Relative Relative luciferase activity CH6953755 0.2

0 50mm 50mm 0 0.03 0.1 0.3 1 CH6953755 (mM) C D CH6953755 (mmol/L) KYSE70 KYSE70_YAP1-5SA #1 KYSE70 1.6 KYSE70_YAP1-5SA #7 KYSE70_YAP1-5SA #1

100 1.4 n.s. KYSE70_YAP1-5SA #7

1.2 n.s. 80 n.s. n.s. n.s. 1 n.s. *** 60 0.8 *** 0.6 *** 40

*** (%) viability Cell 0.4 *** 20

Relative Relative luciferase activity 0.2

0 0 0 0.1 0.4 1 0.001 0.01 0.1 1 CH6953755CH3755 (uM)(mmol /L) CH6953755CH3755 (mmol (uM)/L) Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

YES1 is a targetable oncogene in cancers harboring YES1 gene amplification

Natsuki Hamanaka, Yoshito Nakanishi, Takakazu Mizuno, et al.

Cancer Res Published OnlineFirst August 7, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-3376

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/07/27/0008-5472.CAN-18-3376.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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