YES1 Is a Targetable Oncogene in Cancers Harboring YES1 Gene Amplification

Published OnlineFirst August 7, 2019; DOI: 10.1158/0008-5472.CAN-18-3376 Cancer Molecular Cell Biology Research

YES1 Is a Targetable Oncogene in Cancers Harboring YES1 Gene Ampliﬁcation Natsuki Hamanaka, Yoshito Nakanishi, Takakazu Mizuno, Kana Horiguchi-Takei, Nukinori Akiyama, Hiromi Tanimura, Masami Hasegawa, Yasuko Satoh, Yukako Tachibana, Toshihiko Fujii, Kiyoaki Sakata, Kiyomoto Ogasawara, Hirosato Ebiike, Hiroshi Koyano, Haruhiko Sato, Nobuya Ishii, and Toshiyuki Mio

Abstract

Targeting genetic alterations of oncogenes by molecular- antitumor activity against YES1-amplified cancers in vitro targeted agents (MTA) is an effective approach for treating and in vivo. Yes-associated protein 1 (YAP1) played a role cancer. However, there are still no clinical MTA options for downstream of YES1 and contributed to the growth of YES1- many cancers, including esophageal cancer. We used a short amplified cancers. YES1 regulated YAP1 transcription activ- hairpin RNA library to screen for a new oncogene in the ity by controlling its nuclear translocation and serine phos- esophageal cancer cell line KYSE70 and identified phorylation. These findings indicate that the regulation of YES proto-oncogene 1 (YES1) as having a significant impact YAP1 by YES1 plays an important role in YES1-amplified on tumor growth. An analysis of clinical samples showed cancers and that CH6953755 has therapeutic potential in that YES1 gene amplification existed not only in esophageal such cancers. cancer but also in lung, head and neck, bladder, and other cancers, indicating that YES1 would be an attractive Significance: These findings identify the SRC family kinase target for a cancer drug. Because there is no effective YES1 YES1 as a targetable oncogene in esophageal cancer and inhibitor so far, we generated a YES1 kinase inhibitor, describe a new inhibitor for YES1 that has potential for CH6953755. YES1 kinase inhibition by CH6953755 led to clinical utility.

Introduction survival rate is less than 20% and clinical MTA options are few, especially for the squamous type (2). Some cancers depend on a single dominant oncogene for SRC, a nonreceptor protein tyrosine kinase, has been identified proliferation and survival, a phenomenon called "oncogene as being encoded by a proto-oncogene, and the nine members of addiction" (1). In recent years, some patients with cancers that the SRC family, which includes YES1, SRC, FYN, LYN, and LCK, feature oncogene addiction have greatly benefited from various have various important cellular functions, such as cell growth, molecular-targeted agents (MTA). Examples of successful MTAs adhesion, survival, and differentiation (3). Reports of genetic include anaplastic lymphoma kinase (ALK) inhibitor alectinib for alterations in the SRC family are few; however, gene amplification ALK fusion cancer, EGFR inhibitor erlotinib for EGFR-mutant of the site containing YES1 has been reported in clinic (4, 5). cancer, anti-HER2 antibody trastuzumab for HER2 gene– Furthermore, a recent study found that YES1 gene amplification amplified cancer, and v-raf murine sarcoma viral oncogene homo- was a key mechanism of resistance to EGFR or HER2 inhibi- log B (BRAF) inhibitor vemurafenib for BRAF-mutant cancer (1). tors (6, 7). Although the transforming ability of YES1 has yet to be These MTAs are dramatically effective in cancers with genetic proved, there have been various reports of an association between alterations in the target gene, and their success illustrates why it YES1 and tumorigenicity (8). For example, down-regulating YES1 is important to characterize the cancer type and match the by short hairpin RNA (shRNA) significantly inhibited cell growth treatment to it. Various MTAs are already approved; however, in several malignancies, including colon carcinoma, rhabdomyo- many cancers with high unmet medical needs still have no clinical sarcoma, and basal-like breast cancer (9–11), and some miRNAs MTA options. Esophageal cancer is such a cancer; the 5-year were shown to regulate tumor progression via YES1 regulation (12). Taken together, these reports suggest YES1 may play Research Division, Chugai Pharmaceutical Co., Ltd., Kamakura, Kanagawa, a key role in cancer progression. Japan. Furthermore, there are reports of YES1 participating in numer- ous signaling pathways. Various mechanisms, including interac- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). tions with receptor tyrosine kinases, activate YES1, which in turn activates individual substrates (focal adhesion kinase, BCAR1, Corresponding Author: Yoshito Nakanishi, Chugai Pharmaceutical Co., Ltd., 200 and paxillin, etc.), resulting in the production of factors involved Kajiwara, Kamakura, Kanagawa 247-8530, Japan. Phone: 81-70-4314-8769; Fax: 81-4-6746-5320; E-mail: [email protected] in growth, survival, invasion, and metastasis (3). In this way, YES1 signaling has been roughly explained, but not fully investigated. Cancer Res 2019;XX:XX–XX Some studies reported that YES1 is associated with YAP1 (13, 14). doi: 10.1158/0008-5472.CAN-18-3376 YAP1, originally isolated as a protein that binds to the SH3 2019 American Association for Cancer Research. domain of c-Yes (15), is known as a transcriptional coactivator

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in the Hippo pathway and is reported in many studies to be (System Biosciences). KYSE70, OACP4 C, and K562 were infected involved in tumor progression (16, 17). YAP1 is dysregulated in with lentiviruses containing YES1 wild-type (YES1-WT) or mutant tumors and its functions include disruption of contact inhibition, T348I (YES1-GK) prepared using pEZ-Lv105 (GeneCopoeia). tumor progression, suppression of apoptosis, and release from Cells stably expressing the above genes were established through senescence. The activity of YAP1 is downregulated by phosphor- tolerance to puromycin following the lentivirus infection. ylation of five HxRxxS consensus motifs by the large tumor suppressor (LATS) kinase. Serine-phosphorylated YAP1 localizes shRNA library screening and data analysis to cytoplasm and is degraded by proteasomes (16, 18). Tyrosine- We used Custom Lentiviral shRNA library DECIPHER Human phosphorylated YAP1 by YES1, SRC, LCK, or Abelson murine Module 1, DECIPHER Human Module 2, and DECIPHER Human leukemia viral oncogene homolog (c-ABL) kinases is also Module 3 (Cellecta). Each module contains approximately 27,500 reported, but its function seems context-dependent and has not shRNAs targeting approximately 5,000 genes, with 5–6 shRNAs been fully investigated (13, 14, 19–21). targeting each gene. Further details of the protocol are in Supple- Although selective inhibitors to the SRC family kinases (SFK) mentary Materials and Methods. NGS data were analyzed using have not yet made their way into clinic, dasatinib and bosutinib the Redundant siRNA Activity algorithm (24), which calculates are well known as SFK/multikinase inhibitors. These inhibitors gene-centric P values. We defined P < 0.05 as a screening hit. We also inhibit ABL, which accounts for their approval for the used four genes, PLK1, RBX1, KIF11, and EIF3A, as essential genes treatment of Philadelphia chromosome-positive chronic myeloid for positive controls, and luciferase for a negative control. leukemia. Although these inhibitors can kill various types of solid tumor in preclinical models, their off-target toxicity prohibits Immunoprecipitation of YES1 their use as therapeutics (3, 22). Therefore, no SFK inhibitors have Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology) so far been approved to treat solid tumors. supplemented with cOomplete and PhosSTOP (Roche). To In this study, we conducted a shRNA library screening and immunoprecipitate the YES1 proteins, Dynabeads Protein G identified YES1 kinase as an attractive target among the amplified (Invitrogen) conjugated to anti-YES1 antibody (Wako, 013- 14261) was added to cell lysate and incubated for 2 hours at genes in esophageal cancer. We proved the oncogenic activity of YES1, displayed the YES1 gene amplification in clinical, generated 4 C. Precipitates were washed three times in the wash buffer, an inhibitor that was highly selective against SFK and especially followed by denaturing with the sample buffer solution contain- against YES1, and examined its potential efficacy to inhibit YES1 ing a reducing agent for SDS-PAGE (Life Technologies). The kinase in YES1 gene–amplified tumors. We also evaluated the samples were then subjected to SDS-PAGE and analyzed by association of YES1 with YAP1 as a key factor in YES1 gene– Western blotting. amplified cancers and found that YES1 kinase activity regulated Western blot analysis YAP1 activity by nuclear translocation and serine phosphoryla- Western blot analysis was performed as described previous- tion, and that YAP1 transcriptional activity contributed to the ly (25, 26). The details of the antibodies are in Supplementary proliferation of YES1 gene–amplified cancers. In summary, we Materials and Methods. propose that a YES1 inhibitor has therapeutic potential for YES1 – fi gene ampli ed cancers. Protein kinase assay The inhibitory activity against each kinase was evaluated as Materials and Methods described previously (27). The details of the recombinant proteins are in Supplementary Materials and Methods. Cell lines and reagents Cell lines were obtained from the ATCC, Asterand, Deutsche Cell viability assay Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Cells were seeded in 96-well plates and incubated at 37C European Collection of Authenticated Cell Cultures, Immuno- with inhibitors. After 4 or 7 days, cell viability was measured Biological Laboratories, NCI, RIKEN Bio Resource Center, Health using Cell Counting Kit-8 Solution (Dojindo Laboratories) or Science Research Resources Bank, Health Protection Agency Cul- CellTiter-Glo Assay (Promega). ture Collections, Korean Cell Line Bank, DS Pharma Biomedical, and the Japanese Collection of Research Bioresources (JCRB) Cell Mouse xenograft study Bank. Esophageal cancer cell lines KYSE70, KYSE140, KYSE-180, The mouse xenograft study was performed as described KYSE270, KYSE-410, KYSE-450, and KYSE-510 were kindly pro- previously (28). All in vivo studies were approved by the vided by Dr. Yutaka Shimada (Toyama University, Toyama, Chugai Institutional Animal Care and Use Committee. Female nu Japan) via DSMZ or JCRB (23). All cell lines were obtained more BALB/c-nu/nu mice (CAnN.Cg-Foxn1 /CrlCrlj) were obtained than 1 year prior to experiments and were propagated for less than from Charles River Laboratories and kept under specified 6 months after thawing. All cell lines were cultured according to pathogen-free conditions. Cells (5 106 to 7 106) were sus- the manufacturer's instructions and confirmed as Mycoplasma pended in 200 mLHank'sBalancedSaltSolutionandinjected negative by culture or PCR methods described elsewhere. Cellular subcutaneously into the right flank of the mice. For the Rat-2_mock experiments were performed within 20 passages after thawing. and Rat-2_YES1 models, an equal amount of Matrigel (Corning) was mixed with the cell suspension. Tumor size was measured Generations of Rat-2/KYSE70/OACP4 C/K562 stable using a gauge twice per week, and tumor volume (TV) was cal- transductants culated using the following formula: TV ¼ ab2/2, where a is Rat-2 cells (Human science) were infected with an empty the length of the tumor and b is the width. Once the tumors lentivirus (Rat-2_mock) or lentivirus containing YES1 wild- reached a volume of approximately 200–300 mm3, animals were type (Rat-2_YES1) prepared using pCDH-EF1-MCS-IRES-Puro randomized into groups (n ¼ 3, 4, or 5 in each group),

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Effective YES1-YAP1 Blockade on YES1 Gene–Ampliﬁed Cancers

and treatment was initiated. Compound was orally administered (Rat-2_mock) or a lentiviral vector expressing YES1 (Rat-2_YES1). once a day. The use of a normal fibroblast cell line to validate oncogenic activity is a common procedure for validating transformation Luciferase reporter assay of oncogenes such as ALK fusion, ROS fusion, and FGFR Cells were cotransfected with TEAD1 luciferase reporter plas- fusion (28–30). We used the YES1-transduced bulk cells and mid (pTEAD1-Luc, Signosis) and YAP-irresponsive TK Renilla checked YES1 expression in Rat-2_YES1 cells in vitro and in vivo luciferase reporter (pGL4.74-hRluc-TK, Promega) as a reference (Fig. 1B and C). We observed that YES1 overexpression promoted by using ViaFect (Promega). After 24 hours, cells were treated with tumorigenicity in Rat-2_YES1 cells compared with the Rat-2_mock the compound for another 24 hours. Cell lysates were analyzed cells (Fig. 1D), and Rat-2_YES1 tumors were about 13 times larger for firefly and Renilla luciferase activities with the Dual-Glo Assay than Rat-2_mock tumors at 45 days after inoculation (Fig. 1E), System (Promega). Firefly luciferase activity was normalized by suggesting that YES1 has oncogenic potential in vivo. calculating the ratio to Renilla luciferase activity. The relative luciferase activity was reported as a fold change to the com- YES1 gene amplification occurs in various cancer types pound-untreated control. To evaluate the frequency of YES1 genetic alterations in clinical, we analyzed copy number alterations and mutations in Quantitative real-time PCR The Cancer Genome Atlas (TCGA) data using cBioPortal (http:// Total RNA was extracted and purified using RNeasy 96 Kit www.cbioportal.org/; refs. 31, 32) and found YES1 gene ampli- (Qiagen) and cDNA was synthesized using Transcriptor Uni- fication in various tumor types (Table 1), but because the bio- versal cDNA Master (Roche) according to the manufacturer's logical function of each YES1 mutation is unknown, we did not instructions. Real-time PCR analysis was run on the LightCycler analyze them in detail. We confirmed YES1 gene amplification by 480 System (Roche). For data analysis, counts were normalized FISH in a tissue microarray of esophageal cancer and lung cancer to the housekeeping gene GAPDH at the same condition. (Supplementary Fig. S1A). Next, we used the PrognoScan data- Counts were reported as fold change relative to the untreated base (33) to analyze the correlation between YES1 expression and control. Details of the PCR primers are in Supplementary prognosis for these tumor types. In esophageal and lung cancer, Materials and Methods. the prognosis for patients with high YES1 expression was worse than for those with low YES1 expression at a level that is statis- Immunofluorescence tically significant, suggesting the contribution of YES1 in these Cells were cultured on coverslips or 96-well plates. Cells were malignancies (Supplementary Fig. S1B). Taken together, we fixed, permeabilized, and blocked by Image-iT Fixation/Permea- hypothesized that YES1 gene amplification accompanied with bilization Kit (Life Technologies). Then cells were incubated with high expression could be an anticancer target, not only for YAP1 antibody (Abcam, ab52771) at room temperature for 60 esophageal cancer but also for other cancers, and we set about minutes. The secondary antibody was goat anti-Rabbit IgG cou- generating a YES1 inhibitor. pled with Alexa Fluor 88 (green; Life Technologies) incubated at room temperature for 60 minutes in the dark. Nuclei were YES1 kinase inhibitory activity of CH6953755 counterstained with Hoechst 33342. Images were acquired To obtain a YES1 inhibitor, we performed a high-throughput by immunofluorescence microscopy (Nikon, E-600 Eclipse or screening of a chemical library with more than half-a-million Thermo Fisher Scientific Inc., Cellomics ArrayScan VTI System). compounds at Chugai Pharmaceutical Co. Ltd. and identified a lead compound that inhibited a relatively broad range of kinases but had a different chemical scaffold from known SFK inhibitors. Results We then structurally modified and improved the pharmacokinet- YES1 has an oncogenic role ics profile and selectivity against SFKs, especially YES1. Finally, we In a shRNA library of 15,000 genes, we set out to identify an generated an aminopyrazole derivative, CH6953755, as a potent oncogene with a genetic alteration in esophageal cancer, by and orally available YES1 inhibitor (Fig. 2A; ref. 34). screening the KYSE70 esophageal cancer cell line, using HCT- To investigate the selectivity of CH6953755 and compare it 116 (colorectal cancer) and NCI-H2009 (lung cancer) cell lines with other known SFK inhibitors, dasatinib (35) and bosuti- as controls. In the first trial, which compared KYSE70 with nib (36), we evaluated the inhibitory activity against 39 kinases HCT-116, the number of genes with a significant impact on in an enzyme assay (Fig. 2A and B; Supplementary Table S1). growth found only in KYSE70 was 488. In the second trial, The IC50 value of CH6953755 on the enzyme activity of YES1 which compared KYSE70 with NCI-H2009, the number found was 1.8 nmol/L. Although bosutinib had similar IC50 value as only in KYSE70 was 1,164. There were 128 genes common to CH6953755 and dasatinib had about 10-fold higher activity both trials. We screened genetic alterations of the 128 genes in on YES1 kinase inhibition, CH6953755 exhibited more spec- the Cancer Cell Line Encyclopedia (CCLE) database (https:// ificity for YES1 among the tested SFKs compared with both portals.broadinstitute.org/ccle) and identified only one with compounds (Fig. 2B and C; Supplementary Table S1). We used gene amplification, YES1 (Fig. 1A), which suggested that YES1 dasatinib for a further comparison study of CH6953755 based could be a target molecule for esophageal cancer therapy. on its potent YES1-inhibiting activity but low selectivity. According to an in vitro experiment in a previous publication, To evaluate the kinase selectivity of CH6953755 further, we YES1 had no oncogenic activity (8). However, we proved YES10s used a KINOMEscan Panel (DiscoveRx) consisting of 456 WT and oncogenic activity with in vivo experiments. We lacked access to mutant kinases (Supplementary Table S2). In the panel, esophageal cancer cell lines with YES1 deletion, so we used the CH6953755 at 10 nmol/L bound (at more than 65% inhibition normal rat fibroblast cell line Rat-2 to generate clones that to an ATP analog) to only eight WT kinases, including YES1 and had been stably transduced with an empty lentiviral vector four other SFKs.

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A HCT-116 KYSE70 KYSE70 NCI-H2009

520 243 488 1,164 219 625

KYSE70 KYSE70 360 128 1,036

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 2,400 Rat-2_mock Rat-2_mock Rat-2_YES1 2,000 Rat-2_YES1 ) 3

1,600

1,200

800

Tumor volume (mm 400 *** 0 32 34 36 38 40 42 44 46 Average Average Day after the inoculation 159 㼼 124 mm3 1,574 㼼 619 mm3 (n = 10) (n = 7)

Figure 1. The shRNA library screening identified YES1 as an oncogene for an esophageal cancer cell line. A, Venn diagrams show the number of genes with a significant impact on growth (P < 0.05). The criterion for amplification is an amplification ratio of >2.0. B, Rat-2 parent, Rat-2_mock, and Rat-2_YES1 cells were lysed and analyzed by Western blotting. YES1 proteins were immunoprecipitated (IP) from cell lysates and blotted (IB). C, Xenograft tumors derived from implantation of Rat-2_mock and Rat-2_YES1 cells into mice were lysed and analyzed by Western blot (n ¼ 3 and 4, respectively). YES1 proteins were immunoprecipitated from cell lysates and blotted. D, Rat-2_mock and Rat-2_YES1 cells (5 106) were implanted into nude mice. Tumor volumes in each group were measured. Data are shown as mean SD (n ¼ 10 and 7). Two-tailed Student t test: , P < 0.001, versus Rat-2_mock at final day. E, Representative images of Rat-2_mock and Rat- 2_YES1 mice. Tumor volumes in each group were measured at day 45 after inoculation (n ¼ 10 and 7).

YES1 kinase inhibition leads to cell growth inhibition of YES1- the YES1 expression and copy number data of 1,035 cancer cell amplified cancer cell lines lines in the CCLE database to determine the profile of lines Next, we investigated whether YES1 kinase inhibition inhibited harboring YES1 gene amplification. We defined YES1 gene ampli- the cell growth of YES1-amplified cancer cells. First, we analyzed fication as having a copy number over four. We confirmed that

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Table 1. The clinical prevalence of YES1 gene amplification in various cancer ing that the contributions of SFKs other than YES1 were low in types YES1-amplified cancer cell lines. We focused on SRC as represen- Cancer type Frequency tative of other SFKs and confirmed this with a cell growth Esophageal squamous cell carcinoma (TCGA, 6.3% (6/96 cases) inhibition assay using KYSE70 clones stably transduced with an provisional) Esophageal adenocarcinoma (TCGA, provisional) 5.7% (5/88 cases) empty lentivirus or lentivirus-expressing SRC-WT or YES1-WT Head and neck squamous cell carcinoma (TCGA, 5.1% (27/530 cases) (KYSE70_mock, KYSE70_SRC-WT, and KYSE70_YES1-WT) trea- provisional) ted with CH6953755 or dasatinib. Efficacy against KYSE70_mock Lung squamous cell carcinoma (TCGA, provisional) 4.5% (23/511 cases) and KYSE_YES1-WT was almost the same for treatment with Bladder urothelial carcinoma (TCGA, provisional) 4.4% (18/413 cases) CH6953755 and dasatinib, but the efficacy of CH6953755 in Sarcoma (TCGA, provisional) 3.0% (8/265 cases) KYSE70_SRC-WT was less sensitive than in KYSE70_YES1-WT or Ovarian serous cystadenocarcinoma (TCGA, provisional) 3.0% (18/606 cases) KYSE70_mock, while the efficacy of dasatinib was pretty much NOTE: We analyzed the clinical copy number alteration data using cBioPortal unchanged (Supplementary Table S4). It was thought that SRC (http://www.cbioportal.org/) on August 18, 2018. The results shown here are based upon data generated by the TCGA Research Network (https://cancer initiated activation of the same downstream signal as YES1, which genome.nih.gov/). contributed to cell proliferation when SRC was expressed in KYSE70, and SRC inhibitory activity of CH6953755 was about 20-times weaker than YES1 inhibitory activity, while the SRC YES1 expression was upregulated in amplified cell lines (Supple- inhibitory activity of dasatinib was almost same as YES1 mentary Fig. S2A), and validated this upregulation by measuring (Supplementary Table S1). On the basis of the evidence from YES1 protein levels in four YES1-amplified and four non-YES1– these experiments, we think that YES1 activity is dominant among amplified cancer cell lines (Supplementary Fig. S2B). These results SFKs, and that the cytotoxicity of CH6953755 resulted from confirmed that YES1 gene amplification includes the upregula- YES1 inhibition in YES1-amplified cancer cell lines, all of tion of YES1 expression in YES1-amplified cancer cell lines both in which suggests the therapeutic potential of a YES1 inhibitor for mRNA and protein levels. Next, we assessed the antiproliferative YES1-amplified cancers. activity of YES1 inhibitors. CH6953755, dasatinib, EGFR inhibitor erlotinib, mTOR inhibitor everolimus, and a MEK inhibitor YES1 kinase inhibition suppresses the growth of YES1- CH4987655 (37) were tested against a panel of 66 cancer cell amplified xenograft tumors lines, seven of which had YES1 amplification. YES1-amplified To confirm whether YES1 inhibition led to antitumor activity cancer cell lines showed higher sensitivity to CH6953755 than in vivo, as well as cell growth inhibition in vitro, first we evaluated non-YES1–amplified cancer cell lines (Fig. 3A; Supplementary the in vivo efficacy of YES1 inhibitor CH6953755 in a Rat-2_YES1 Table S3). CH6953755 prevented the autophosphorylation at xenograft mouse model. After oral treatment of CH6953755 at Tyr426 of YES1 that upregulates enzymatic activity in KYSE70 60 mg/kg, we observed antitumor activity (Fig. 4A) accompanied cells harboring YES1 amplification (Fig. 3B). Dasatinib also with phospho-Tyr426 YES1 suppression in xenograft tumors showed more potent efficacy against YES1-amplified cell lines (Fig. 4B). Next, we utilized native cancer cell lines harboring than against nonamplified lines (Supplementary Fig. S3A), but YES1 gene amplification. Consistent with the findings in the Rat- YES1 amplification did not seem to predict the efficacy of erlo- 2_YES1 model, CH6953755 showed antitumor activity against tinib, everolimus, or CH4987655 (Supplementary Fig. S3B–S3D, xenograft models with YES1 amplification, such as KYSE70 [copy Supplementary Table S2). Therefore, we considered that YES1 number ¼ 9.9, maximum tumor growth inhibition (TGI) 90% at gene amplification status could predict the efficacy of a YES1 the MTD] and RERF-LC-AI (copy number ¼ 5.1, maximum TGI inhibitor. Next, we tried to check whether the CH6953755 cyto- 93% at the MTD; Fig. 4C). In contrast, CH6953755 did not show toxicity was the result of YES1 inhibition in YES1-amplified cancer antitumor activity against xenograft models with no YES1 cell lines using the commonly used chemical–genetic approach amplification, such as ACHN (copy number ¼ 1.8, maximum of engineering an inhibitor-resistant mutant of YES1 (38, 39). TGI 13% at the MTD) and HARA (copy number ¼ 2.7, max- From YES1-amplified KYSE70 and OACP4 C, and YES1 nonam- imum TGI 13% at the MTD; Fig. 4D). These data were in line plified K562 cell lines, we generated clones stably transduced with with the in vitro observations. We also tested dasatinib against a a lentiviral vector expressing YES1-WT or mutant T348I, a so- YES1-amplified KYSE70 model. Because its toxicity limits the called gatekeeper mutation (YES1-GK) that had a point mutation dose, dasatinib showed moderate efficacy (maximum TGI 41% at the binding site with CH6953755 and was expected to be at the MTD; Supplementary Fig. S6). Then we evaluated the resistant to CH6953755. In KYSE70 cells expressing YES1-GK suppression of YES1 signaling in tumor tissues by conducting (KYSE70_YES1-GK), phosphorylation at Tyr426 was not sup- Western blotting 24 hours after the last administration of daily pressed by CH6953755 treatment, unlike in KYSE70 cells expres- dosing. The blots demonstrated that CH6953755 suppressed sing YES1-WT (KYSE70_YES1-WT; Fig. 3C). As we expected, the phospho-Tyr426 YES1 in a dose-dependent manner (Fig. 4E). two cell lines expressing YES1-GK with amplified YES1 gene These results suggest that YES1 kinase inhibition by became resistant to CH6953755, while this was not the case with CH6953755 leads to selective antitumor activity against the line without amplified YES1 (Fig. 3D). Similar results were YES1-amplified cancers both in vitro and in vivo,andYES1 found with dasatinib (Supplementary Fig. S4). These results kinase plays a crucial role in YES1-amplified cancers. indicated that YES1 kinase inhibition inhibited the cell growth of YES1-amplified cancer cell lines. YAP1 plays a role downstream of YES1 in YES1-amplified cancer Furthermore, we analyzed whether the other SFKs had an effect Next, we investigated how YES1 inhibition achieved antitumor on YES1-amplified cancer cell lines. We examined SFK expression activity, focusing on YAP1. Because genetic alterations in the profiles in YES1-amplified cancer cell lines and determined the Hippo pathway, such as LATS2 gene deletion, result in YAP1 dominance of YES1 expression (Supplementary Fig. S5), suggest- activation that leads to tumor progression (40), we hypothesized

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A CH6953755 Dasatinib Bosutinib

µ µ µ B CH6953755 IC50 ( mol/L) Dasatinib IC50 ( mol/L) Bosutinib IC50 ( mol/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 Figure 2. FGFR2 FGFR2 FGFR2 Compared with dasatinib and JAK2 JAK2 JAK2 bosutinib, CH6953755 tends to be KDR KDR KDR FLT3 FLT3 FLT3 selective to YES1. A, Chemical IRAK1 IRAK1 IRAK1 structures of CH6953755, dasatinib, ALK ALK ALK and bosutinib. B, Kinase inhibitory AXL AXL AXL activity of CH6953755, dasatinib, CHK2 CHK2 CHK2 FAK FAK FAK and bosutinib against 39 kinases. MEK1 MEK1 MEK1 C, Kinase inhibitory activity of CHK1 CHK1 CHK1 CH6953755, dasatinib, and PKC alpha PKC alpha PKC alpha ﬁ PKC beta1 PKC beta1 PKC beta1 bosutinib against ve SFKs. 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 50 16 50 16 16 14 14 14 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Ratio to IC to Ratio YES1 Ratio to IC to Ratio YES1 2 2 IC to Ratio YES1 2 0 0 0

that YES1 gene amplification is a noncanonical YAP1 activation pression was derived from YES1 kinase inhibition, we utilized mechanism in cancer cells. The transcription factor TEAD has KYSE70_YES1-GK and found that CH6953755 could not sup- been reported to bind YAP1 and play a major role in YAP1 press their TEAD luciferase reporter activity, even though it oncogenic function (41). YAP1 functions as a transcriptional could suppress the activity in KYSE70_YES1-WT (Fig. 5B). coactivator for TEAD to induce gene expression, such as Furthermore, CH6953755 suppressed the expression of YAP1 ANKRD1 and CYR61, thereby promoting cell growth, prolifer- downstream genes, ANKRD1 and CYR61, in KYSE70_ YES1- ation, and survival (16, 42). Therefore, we transfected the cell WT, without affecting the YAP1 expression level, and this lines with TEAD luciferase reporter and tested the effect of YES1 suppression was abrogated in KYSE70_ YES1-GK (Fig. 5C). inhibitor, CH6953755, on luciferase activity. CH6953755 sup- These phenomena were also observed with dasatinib treatment pressed TEAD luciferase reporter activity in YES1-amplified (Supplementary Fig. S7A and S7B), suggesting that YES1 kinase KYSE70 and RERF-LC-AI (Fig. 5A). To confirm that the sup- activity regulates YAP1 activity.

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A B C

100 KYSE70_ KYSE70_ YES1-WT YES1-GK

10 CH6953755 CH6953755 (µmol/L) (µmol/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 input 1 ( µ mol/L) IB: pY416 SFK IB: pY416 SFK 50

IC 0.1 IB: YES1 IB: YES1 input

0.01 IB: GAPDH IB: GAPDH

YES1 amp YES1 non-amp

D KYSE70 OACP4 C K562 120 (YES1 amp) (YES1 amp) 120 (YES1 non-amp) 120 100 100 100 80 80 YES1-WT 80 60 60 YES1-GK 60

40 40 40 Cell viability (%) Cell viability (%) Cell viability (%)

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 (µmol/L) Concentration (µmol/L) Concentration (µmol/L)

Figure 3.

YES1 kinase inhibition inhibits the cell growth of YES1-amplified cancer cell lines. A, The IC50 value of CH6953755 was evaluated in a cell proliferation assay. In total, seven YES1-amplified and 59 non-YES1–amplified cancer cell lines were analyzed. Two-tailed Student t test: , P < 0.001. B and C, The indicated concentrations of CH6953755 were applied for 2 hours to the KYSE70 cell line (B) and cloned cell lines, KYSE70_YES1-WT and KYSE70_YES1-GK (C). The cells were lysed and analyzed by Western blot. YES1 proteins were immunoprecipitated (IP) from cell lysates and blotted (IB). D, YES1-amplified cancer cell lines KYSE70 and OACP4 C, and non-YES1–amplified cancer cell line K562 expressing YES1-WT or YES1-GK were seeded in 96-well plates and treated for 4 days with various concentrations of CH6953755. Subsequently, cell viability was measured.

YES1 regulates YAP1 activity by controlling its nuclear all of the HxRxxS consensus motifs, makes YAP1 constitutively translocation and serine phosphorylation via kinase activity active by abolishing the phosphorylation by LATS (43), so we Our next question was how YES1 regulated YAP1 activity. generated two KYSE70 clones, #1 and #7, which were stably First, we looked at the YAP1 localization. Multikinase inhibitor transduced with a lentiviral vector expressing YAP1 5SA. In dasatinib has been reported to inhibit nuclear localization these clones, the suppression of TEAD luciferase reporter activ- of YAP1 (20, 21), so we hypothesized that YES1 regulated ity after treatment with CH6953755 was abrogated (Fig. 6C), YAP1 activity by controlling YAP1 localization. We evaluated and the antiproliferative activity of CH6953755 was dimin- the localization of YAP1 and confirmed the phenomenon in ished (Fig. 6D). Taken together, the findings suggest that YES1 KYSE70_YES1-WT or -GK after treatment with CH6953755. YAP1 regulates YAP1 activity by serine phosphorylation indirectly nuclear localization was inhibited in the KYSE70 cell line expres- and by nuclear localization. In conclusion, YES1-YAP1 signal sing YES1-WT, and the inhibition was abrogated in KYSE70_ inhibition by CH6953755 leads to antitumor activity in YES1- YES1-GK (Fig. 6A; Supplementary Fig. S8). Furthermore, we amplified cancer cells. confirmed this phenomenon in in vivo setting (Supplementary Fig. S9). Next, we evaluated the effects of YAP1 phosphorylation sites on CH6953755 activity. Prior work has demonstrated that Discussion YES1 phosphorylation of YAP1 at the tyrosine 357 site regulated In this study, first, we screened a shRNA library to identify YES1 YAP1 transcriptional activity (13), so we examined the TEAD as a fascinating oncogenic target, and we also analyzed the Project luciferase reporter activity when the following YAP1 mutations Achilles data (44). The frequency of YES1 gene amplification were being expressed: Y357E, which is phosphomimetic, or among cell lines in Project Achilles is 2.6% (13/502). We used Y357F, which is phosphodead (19). Neither the YAP1 Y357E nor four genes, PLK1, RBX1, KIF11, and EIF3A, as essential genes the Y357F mutation affected the suppression of YAP1 transcrip- for positive control, but these genes showed no significance in tional activity by CH6953755 (Fig. 6B), which suggested that YES1-amplified cancer cell lines based on our analysis of Achilles phosphorylation of YAP1 at tyrosine 357 did not have any impact data. Next, we changed the criteria for positive control by using on the control of YAP1 activity by YES1 in YES1-amplified cancer. the average of the most potent three of the four individual Then we assessed the effect of YAP1 serine sites, which are EIF3A shRNAs to show significance (P < 0.05). Four of 13 important for YAP1 regulation in the canonical Hippo pathway. YES1-amplified cancer cell lines met this criterion, but we did Several groups have reported that YAP1 5SA, which is mutated in not identify either YES1 or YAP1 as hits. This result differs from

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A 1,600 B Vehicle CH6953755 1,400 )

3 60 mg/kg Vehicle 60 mg/kg 1,200 IP: YES1 1,000 IB: pY416 SFK 800

600 IB: YES1

400 Tumor volume (mm 200 * IB: YES1 input 0 333129 35 3937 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 1,400 450 900 800 400 800 ) ) 1,200 3 3 700 350 700 1,000 n.s. 600 300 600 n.s. 500 800 250 500 * n.s. 400 600 200 400 300 *** * 150 300 *** 400 200 *** ** 100 200 Tumor volume (mm Tumor volume (mm ** 100 200 50 100 0 0 0 0 23211917151311 19 3431282522 9 11 13 15 17 19 21 23 26242220181614

120 120 120 120

100 100 100 100

80 80 80 80 Body weight (%) Body weight (%) weight Body 23211917151311 252219 343128 9 11 13 15 17 19 21 23 26242220181614 Day after the inoculationDay after the inoculation Day after the inoculation Day after the inoculation

7.5 mg/kg Vehicle Vehicle 60 mg/kg 15 mg/kg 30 mg/kg 60 mg/kg

CH6953755 CH6953755 E (mg/kg) (mg/kg) Vehicle 7.5 15 30 60 Vehicle 7.5 15 30 60 YES1 IB: pY416 SFK IP: IB: YES1 input IB: GAPDH KYSE70 RERF-LC-AI

Figure 4. CH6953755 shows selective antitumor activity against YES1-amplified xenograft tumors. Mice bearing Rat-2_YES1 cells were treated with CH6953755 60 mg/kg or vehicle orally once daily for 10 days. A, Tumor volume in each group was measured. Data are shown as mean SD (n ¼ 3). Two-tailed Student t test: , P < 0.05, versus vehicle treatment at final day. B, Xenograft tumors were extracted 24 hours after the last dosing and analyzed by Western blotting (n ¼ 3). YES1 proteins were immunoprecipitated (IP) from cell lysates and blotted (IB). C–E, CH6953755 at the indicated doses was given orally once daily to xenograft models bearing cells with YES1 amplification, KYSE70 (for 11 days) and RERF-LC-AI (for 14 days; C) or xenograft models bearing cells without YES1 amplification, ACHN (for 12 days) and HARA (for 11 days; D). Tumor volume and body weight change in each group were measured. Data are shown as mean SD (n ¼ 4). C, Parametric Dunnett test: , P < 0.05; , P < 0.01; , P < 0.001; n.s., no significant difference, versus vehicle treatment at final day. D, Two-tailed Student t test: n.s., no significant difference, versus vehicle treatment at final day. E, Xenograft tumors of KYSE70 (n ¼ 4 and 3) and RERF-LC-AI (n ¼ 4 and 2) were extracted 24 hours after the last dosing and analyzed by Western blotting. YES1 proteins were immunoprecipitated from cell lysates and blotted. CN, copy number.

our shRNA screening result, but we think that this is due to the than KYSE70. We determined that we could conﬁrm our shRNA different experimental and analytic conditions, as well as the screening result showing that YES1 had an important role in possibility of lower silencing efﬁciency of YES1 shRNAs and YES1 KYSE70 by using the chemical–genetic approach of engineering copy number in the analyzed cell lines with a lower copy number an inhibitor-resistant GK mutant of YES1 (Fig. 3D).

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Effective YES1-YAP1 Blockade on YES1 Gene–Ampliﬁed Cancers

A B KYSE70_YES1-WT KYSE70 RERF-LC-AI KYSE70_YES1-GK 1.6 1.2 1.2 n.s. 1.4 1 1 n.s. 1.2 n.s. n.s. n.s. 0.8 0.8 1

0.6 0.6 * 0.8 *** *** ** 0.6 0.4 *** *** 0.4 0.4 *** 0.2 0.2 *** *** *** 0.2 Relative luciferase activity Relative luciferase activity Relative luciferase activity 0 0 0 310.30.10 310.30.10 0.10 310.3 CH6953755 (µmol/L) CH6953755 (µmol/L) CH6953755 (µmol/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. 1 n.s. n.s. 1 1 n.s. ** *** n.s. 0.8 0.8 0.8 *** 0.6 0.6 0.6 Relative mRNA Relative mRNA ***

0.4 0.4 Relative mRNA 0.4 *** *** 0.2 0.2 0.2

0 0 0 0 0.001 0.01 10.1 0 0.001 0.01 10.1 0 0.001 0.01 10.1 CH6953755 (µmol/L) CH6953755 (µmol/L) CH6953755 (µmol/L)

KYSE70_YES1-WT KYSE70_YES1-WT KYSE70_YES1-WT KYSE70_YES1-GK KYSE70_YES1-GK KYSE70_YES1-GK

Figure 5. YAP1 plays a role downstream of YES1 in YES1-ampliﬁed cancer. The effect of a YES1 inhibitor on luciferase activity was tested in KYSE70 and RERF-C-AI (A), and KYSE70_YES1-WT and KYSE70_YES1-GK (B) cells. Cells were cotransfected with TEAD1 luciferase and Renilla luciferase reporters. After 24 hours, cells were treated with the indicated concentrations of CH6953755 for 24 hours, and the relative luciferase activity was determined. Data are shown as mean SD (n ¼ 3). C, KYSE70_YES1-WT and KYSE70_YES1-GK were treated with the indicated concentrations of CH6953755 for 24 hours. Gene expression levels were determined by quantitative PCR. Data are shown as mean SD (n ¼ 3). Parametric Dunnett test: , P < 0.05; , P < 0.01; , P < 0.001; n.s., no signiﬁcant difference, versus DMSO control treatment for each cell line.

We demonstrated that YES1 has oncogenic potential and is The YES1 gene might also be enriched in patients insensitive to activated by gene amplification in several tumor types. There- several current standards of care. One study reported that treat- fore, patients with the amplified YES1 gene are expected to ment with 5-fluorouracil (5-FU) upregulated YES1 and YAP1 in benefit from a YES1 inhibitor. At present, the well-known patients with colorectal cancer and these patients have a poor SFK/multikinase inhibitors dasatinib and bosutinib are not prognosis (47). Treatment with 5-FU was also reported to increase effective against solid tumors in clinical. One often-cited the gene copy number of the region that includes thymidylate reason is that there are no prognostic biomarkers related to synthetase (TYMS), a target of 5-FU, in patients with colorectal SFK activity that can be used to select patients for clinical cancer, and this increase is considered to be one of the mechan- trials (3, 22). However, even if an appropriate biomarker was isms of 5-FU resistance (48). As the chromosome locus of TYMS is found, the dose regimen for chronic myeloid leukemia is so close to YES1, the YES1 gene could be amplified together with insufficient to inhibit phosphorylation of SRC and, moreover, the TYMS gene. Because 5-FU is the first-line therapy in esoph- is close to the MTD (45, 46). Consistent with the finding in ageal cancer, this pattern of resistance could also happen there. clinical, dasatinib even at the MTD achieved moderate in vivo Although patients with esophageal cancer in general have quite a efficacy against YES1-amplified KYSE70 xenograft model poor prognosis, the segment of patients with high YES1 expres- (Supplementary Fig. S6), whereas treatment with CH6953755 sion has a worse prognosis than the segment with low expression achieves significant efficacy at a nontoxic dose (Fig. 4C). (Supplementary Fig. S1B). Furthermore, YES1 amplification is a These data suggest that the selectivity of CH6953755 will mechanism of resistance to some MTAs (6, 7). For example, allow the dose in clinic to be increased to a level sufficient to acquired amplification of YES1 is a recurrent mechanism of suppress YES1 kinase and that CH6953755 could be effective resistance to EGFR inhibition in EGFR-mutant lung cancers for in meeting the high unmet medical needs of patients with which YES1 inhibition is effective (49). Moreover, reports show YES1-amplified cancer. that YAP1 is essential for the suppression of antitumor

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A KYSE70_ KYSE70_ B YES1-WT YES1-GK 1.2 YAP1_WT 1 n.s. YAP1_Y357F n.s. DMSO YAP1_Y357E * 0.8 n.s. n.s. 50 µm 50 µm 0.6 n.s. n.s. n.s. 0.4

CH6953755 Relative luciferase activity 0.2

0 50 µm 50 µm 0 0.03 0.1 0.3 1 CH6953755 (µmol/L)

C KYSE70 D 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. n.s. n.s. n.s. 80 1 n.s. *** 60 0.8 *** 0.6 *** 40 *** *** 0.4 Cell viability (%) 20 0.2 Relative luciferase activity 0 0 0 0.1 0.4 1 0.001 0.01 0.1 1 CH6953755 (µmol/L) CH6953755 (µmol/L)

Figure 6. YES1 regulates YAP1 activity by controlling its nuclear translocation and serine phosphorylation. A, KYSE70_YES1-WT and KYSE70_YES1-GK cells were treated with CH6953755 1 mmol/L for 24 hours. The subcellular location of YAP1 was determined by immunofluorescence. High magnification of the area marked within the frame is in Supplementary Fig. S8. B, KYSE70 cells were cotransfected with TEAD1-luciferase and Renilla luciferase reporters with YAP1_WT, YAP1_Y357F, or YAP1_Y357E plasmids. After 24 hours, cells were treated with the indicated concentrations of CH6953755 for 24 hours, and the relative luciferase activity was determined. Data are shown as mean SD (n ¼ 3). Parametric Dunnett test: , P < 0.05; n.s., no significant difference, versus YAP1_WT for each compound concentration. C, KYSE70, KYSE70_YAP1-5SA #1, and KYSE70_YAP1-5SA #7 cells were cotransfected with TEAD1-luciferase and Renilla luciferase reporters. After 24 hours, cells were treated with the indicated concentrations of CH6953755 for 24 hours, and the relative luciferase activity was determined. Data are shown as mean SD (n ¼ 3). Parametric Dunnett test: , P < 0.001; n.s., no significant difference, versus DMSO control treatment for each cell line. D, KYSE70, KYSE70_YAP1-5SA #1, and KYSE70_YAP1-5SA #7 cells were seeded in 96-well plates and treated for 4 days with various concentrations of CH6953755. Subsequently, cell viability was measured. Data are shown as mean SD (n ¼ 3). Parametric Dunnett test: , P < 0.001, versus KYSE70 cell at final day.

immunity (50). These lines of evidence suggest the presence of YAP1 activity? Are there other important YES1 downstream YES1 gene amplification in groups that are insensitive to chemo- signals in YES1-amplified cancers? Regarding YAP1 regulation therapy and MTAs, also suggesting that YES1-amplified cancers by YES1, previous research found that YES1 directly phosphor- are not sensitive to immune checkpoint inhibitors. Therefore, we ylated YAP1 at tyrosine 357 and regulated YAP1 transcriptional think that CH6953755 will provide benefit to these segments. activity in b-catenin–driven cancers (13). In contrast to previous We revealed that YES1 regulates YAP1 activity by controlling research, our evaluation of YES1-amplified cancer expressing YAP1 localization, and that the YES1-YAP1 signal is important in phosphomimetic Y357E and phosphodead Y357F showed that the proliferation of YES1-amplified cancers. In the cell growth tyrosine 357 phosphorylation did not contribute to YAP1 tran- inhibition assay, the YES1-amplified cell line KYSE-510 was scriptional activity (Fig. 6B). On the other hand, our evaluation in insensitive to CH6953755 and dasatinib (Supplementary YES1-amplified cancer expressing YES1-GK proved that YES1 Table S3), and harbored not only YES1 gene amplification but kinase activity is indispensable for YAP1 transcriptional activity also YAP1 gene amplification. This suggests that YES1 could exist and nuclear translocation (Figs. 5B and C and 6A), and the upstream of YAP1, which also supports our findings. However, evaluation in YES1-amplifed cancer expressing five serine- two points are still unclear: how precisely does YES1 regulate mutated YAP1 implies that YAP1 serine phosphorylation has an

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Effective YES1-YAP1 Blockade on YES1 Gene–Ampliﬁed Cancers

effect on the YES1-YAP1 signal (Fig. 6C and D). These results Acquisition of data (provided animals, acquired and managed patients, suggest that YES1 may indirectly activate YAP1 via phosphoryla- provided facilities, etc.): N. Hamanaka, T. Mizuno, K. Horiguchi-Takei, tion of some other kinase involved in the regulation of YAP1 N. Akiyama, H. Tanimura, M. Hasegawa Analysis and interpretation of data (e.g., statistical analysis, biostatistics, serine phosphorylation, which in turn suggests that the mecha- computational analysis): N. Hamanaka, Y. Tachibana, T. Fujii, K. Sakata, nism by which YES1 regulates YAP1 may be dependent on the K. Ogasawara cellular context. As for other YES1 downstream signals important Writing, review, and/or revision of the manuscript: N. Hamanaka, for proliferation, we consider that signals other than YAP1 do Y. Nakanishi, K. Ogasawara, H. Ebiike, H. Koyano, T. Mio contribute to the proliferation of YES1-amplified cancers. In cells Administrative, technical, or material support (i.e., reporting or organizing expressing the YAP1 constitutive active form, YAP1 5SA, suppres- data, constructing databases): Y. Satoh, K. Ogasawara, H. Ebiike, H. Koyano, T. Mio sion of YAP1 transcriptional activity by CH6953755 was abro- Study supervision: N. Hamanaka, Y. Nakanishi, H. Koyano, H. Sato, N. Ishii, gated completely, but the abrogation of cell growth inhibition T. Mio was partial (Fig. 6C and D). Therefore, we think that known YES1 downstream signals, such as MAPK and AKT, or other signals may Acknowledgments be involved in the proliferation of YES1-amplified cancers. We thank Yasue Nagata, Maiko Izawa, Yumiko Hashimoto, Tomoaki In summary, we suggest that YES1-YAP1 signal inhibition is a Hayashi, Yusuke Ide, and Fumie Sawamura for performing pharmacologic mode of action through which CH6953755 achieves efficacy, and assays; Chiaki Senoo, Hiroshi Tanaka, Kenji Kashima, Hironori Mutoh, Yuko Aoki, Masahiro Aoki, and Takuo Tsukuda for helpful discussion; and that CH6953755 has therapeutic potential for patients harboring Hideaki Mizuno for PrognoScan analysis. This study was funded by Chugai YES1 fi the gene ampli cation. Pharmaceutical Co., Ltd.

Disclosure of Potential Conflicts of Interest The costs of publication of this article were defrayed in part by the payment of No potential conflicts of interest were disclosed. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: N. Hamanaka, Y. Nakanishi Received October 26, 2018; revised February 18, 2019; accepted July 23, 2019; Development of methodology: N. Hamanaka, Y. Nakanishi, K. Ogasawara published first August 7, 2019.

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OF12 Cancer Res; 2019 Cancer Research

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

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