MK5 Regulates YAP Stability and Is a Molecular

Published OnlineFirst October 2, 2019; DOI: 10.1158/0008-5472.CAN-19-1339

Cancer Molecular Cell Biology Research

MK5 Regulates YAP Stability and Is a Molecular Target in YAP-Driven Cancers Jimyung Seo1, Min Hwan Kim1,2, Hyowon Hong1, Hyunsoo Cho1, Seongyeol Park1, Sang Kyum Kim3, and Joon Kim1

Abstract

Transcriptional regulator YAP is activated in multiple dation and cytoplasmic retention. Downregulating MK5 human cancers and plays critical roles in tumor initiation, expression inhibited the survival of YAP-activated cancer cell progression, metastasis, and drug resistance. However, thera- lines and mouse xenograft models. MK5 upregulation was peutic targeting of the Hippo–YAP pathway has been chal- associated with high levels of YAP expression and poor prog- lenging due to its low druggability and limited knowledge of nosis in clinical tumor samples, confirming its important role YAP regulation in cancer. Here we present a functional screen for YAP activity in human cancer. These results uncover MK5 as and identify a novel therapeutic target for YAP-driven tumor- a novel factor that regulates YAP stability, and targeting the igenesis. RNAi screening using an oncogenic YAP activation YAP degradation pathway controlled by MK5 is a potential model identified the serine/threonine kinase MK5 as a positive strategy for suppressing YAP activity in cancer. regulator of YAP. MK5 physically interacted with YAP and counteracted CK1d/e-mediated YAP ubiquitination and deg- Significance: These findings reveal MK5 is a novel kinase radation independent of LATS1/2. MK5 kinase activity was that regulates YAP in a LATS-independent manner and can be essential for protecting YAP from ubiquitin-mediated degra- targeted for cancer therapy.

Introduction anticancer agents (2, 3). However, despite intense recent interest, clinical targeting of the Hippo–YAP pathway has been challenging Cancer genome sequencing efforts have uncovered several due to low druggability of the pathway and limited understanding signaling pathways in which major components are frequently of its regulatory mechanisms in human cancer. mutated in human cancers (1). Although robust clinical activity of In the canonical Hippo–YAP pathway, sequential phosphory- EGFR, ALK, and BRAF-targeted drugs has been demonstrated, it is lation of the core Hippo kinases MST and LATS leads to phos- lagging behind to find new effective targets beyond the receptor phorylation of the pathway effector YAP and its paralog TAZ (3). tyrosine kinase pathway for anticancer drug development. Recent Phosphorylation by LATS1/2 inhibits the transcriptional activity studies highlight the association of the Hippo–YAP pathway with of YAP through cytoplasmic sequestration and proteasomal deg- principal cancer features (2, 3). Overactivation of YAP can induce radation. The core Hippo kinases and their adaptors are regarded tumorigenesis, and YAP is also associated with cancer stem cell as tumor suppressors (4). Previous studies uncovered a number of properties, epithelial–mesenchymal transition, metastasis, and regulatory inputs for the Hippo kinases, including GPCRs (5), drug resistance (3). Targeting the Hippo–YAP pathway is expected Rho GTPases (6), the actin cytoskeleton (7), the mevalonate to be beneficial not only for suppressing tumorigenesis and tumor pathway (8), and hypoxia (9). However, the Hippo signaling growth, but also for overcoming metastasis and resistance to pathway does not have a designated ligand–receptor pair, making it difficult to develop a specific agonist for the pathway. Consid- ering that Hippo kinase activity is affected by multiple cellular 1Graduate School of Medical Science and Engineering, Korea Advanced Institute cues, targeting one of the regulatory inputs would result in modest of Science and Technology (KAIST), Daejeon, Korea. 2Division of Medical antitumor activity. Moreover, the expression of the Hippo kinases Oncology, Department of Internal Medicine, Yonsei University College of Med- is frequently blocked by genetic alterations (10, 11). icine, Seoul, Korea. 3Department of Pathology, Yonsei University College of Medicine, Seoul, Korea. Direct inhibition of the binding between YAP and its partner transcription factor TEAD is also challenging because inhibition Note: Supplementary data for this article are available at Cancer Research of the protein–protein interaction is difficult due to flat and large Online (http://cancerres.aacrjournals.org/). interfaces lacking targetable binding pockets (12). Verteporfin, a J. Seo and M.H. Kim contributed equally to this article. YAP antagonist known to interfere with YAP–TEAD complex Corresponding Authors: Joon Kim, Graduate School of Medical Science and formation, has low YAP-inhibitory efficacy (13) and low speci- Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 ficity protein crosslinking activity (14). The hurdles in targeting Daehak-ro, Daejeon 34141, Republic of Korea. Phone: 824-2350-4242; E-mail: the Hippo signaling pathway or YAP–TEAD interaction make it [email protected]; and Sang Kyum Kim, Department of Pathology, Yonsei attractive to target noncanonical YAP-regulatory mechanisms that University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea. Phone: 821-599-1004; E-mail: [email protected] are active in YAP-driven cancers. A number of studies have demonstrated that the activity and stability of YAP can be regu- Cancer Res 2019;79:6139–52 lated by cellular metabolic status (15), osmotic stress (16), and doi: 10.1158/0008-5472.CAN-19-1339 autophagy (17) in a Hippo-independent manner. These Hippo- 2019 American Association for Cancer Research. independent YAP regulatory factors play a complex role in various

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cellular processes, and thus pharmacologic manipulation of these that mimics the context in which the Hippo signaling pathway is factors will induce pleiotropic effects. Therefore, there is a need for inactivated, we edited LATS1 and LATS2 in RPE1 cells using the unbiased screening approach to identify novel regulators of YAP CRISPR-Cas9 system (Supplementary Fig. S1A). Frameshift muta- activity or stability that are good drug targets. tions in both copies of LATS1 (exon 5) and LATS2 (exon 3) were In this study, we performed cell-based RNA interference introduced, and the loss of LATS1/2 expression was confirmed by (RNAi) library screening to identify genes required for nuclear an immunoblot assay (Fig. 1A; Supplementary Table S1). As enrichment of YAP. Our screening identified the serine/threo- expected, YAP phosphorylation was substantially reduced by nine kinase MK5 (also called MAPKAPK5 or PRAK) as a novel LATS1/2 inactivation (Fig. 1A; Supplementary Fig. S1B). Unlike positive regulator of YAP. We found that ubiquitination and parental RPE1 cells, LATS1/2-null RPE1 cells exhibited nuclear degradation of YAP is protected by MK5. Moreover, we provide YAP/TAZ enrichment even at high cell densities (Fig. 1B). YAP/ evidence that MK5 is a useful target molecule for developing TAZ nuclear localization in LATS1/2-null RPE1 cells was not drugs for YAP-driven cancers including uveal melanoma and suppressed by inhibitors of actin polymerization or actomyosin malignant mesothelioma. contraction (Fig. 1C). Transient cotransfection of LATS1 and LATS2 caused YAP/TAZ cytoplasmic retention, confirming that constitutive YAP/TAZ nuclear localization is attributed to the Materials and Methods inactivation of LATS1/2 (Supplementary Fig. S1C). Residual Cell culture YAP phosphorylation at serine 127 (S127) was not reduced by Cells were acquired from ATCC and large frozen stocks were LATS1/2 siRNA transfection (Supplementary Fig. S1D). Along made to ensure against contaminations by other cell lines. All cell with YAP/TAZ nuclear localization, the expression of YAP/TAZ lines were used within 10 passages after reviving from the frozen target genes was upregulated in LATS1/2-null RPE1 cells (Sup- stocks. Cells were free of Mycoplasma contamination as deter- plementary Fig. S1E). mined by staining cells with DAPI every 2 or 3 passages. Cells were Consistent with the fact that the Hippo pathway mediates cultured in the following media: [RPE1 cells: DMEM/F12 (Wel- contact inhibition of cell proliferation, LATS1/2-null cells con- gene); MSTO-211H, H2373, 92.1, and OCM1 cells: RPMI1640 tinued to proliferate at high cell densities (Supplementary (Welgene); SKMEL cells: MEM (Welgene); WM3248 cells: MCDB Fig. S1F). The rate of cell proliferation and the fraction of S/ 153 (Welgene)] supplemented with 10% FBS and 1% penicillin/ G2–M phase cells were also increased in LATS1/2-null context streptomycin. RPE1 cell lines carrying inactivating mutations in (Supplementary Fig. S1G and S1H). We next examined whether both LATS1 and LATS2 genes were established using the CRISPR- LATS1/2 loss resulted in oncogenic transformation of RPE1 cells. Cas9 system according to the previously published protocol (18). Parental RPE1 cells failed to grow in three-dimensional (3D) PLX4032-resistant SKMEL28 and WM3248 cells were established Matrigel culture, whereas LATS1/2-null RPE1 cells were highly according to the previously published protocol (19). proliferative in Matrigel (Supplementary Fig. S1I). Nuclear localization of YAP/TAZ in LATS1/2-null RPE1 cells persisted in Kinome siRNA library screening Matrigel culture (Supplementary Fig. S1J). A transwell migration A kinome-wide siRNA library targeting 607 human kinases assay showed that LATS1/2 loss significantly increased invasion (Dharmacon) was used in this study. Two biologic replicates of capacity of RPE1 cells (Supplementary Fig. S1K). Moreover, in vivo LATS1/2 knockout RPE1 cells were transfected with target siRNAs, tumorigenic potential of LATS1/2-null RPE1 cells was demon- fixed, and stained. Subcellular YAP/TAZ immunofluorescence was strated by xenograft into immunocompromised mice (BALB/c analyzed by CellProfiler software (Broad Institute). Detailed nude). LATS1/2-null RPE1 cells efficiently formed tumors after methods are presented in the Supplementary Data. transplantation, whereas parental RPE1 cells did not develop detectable tumors (Fig. 1D). Hematoxylin and eosin staining of Xenograft the LATS1/2-null RPE1 tumor showed pleomorphism and For xenograft experiments, female nude mice (6 weeks old) increased nuclear-to-cytoplasm ratio, consistent with typical find- were injected subcutaneously with the indicated cells and the ings of carcinoma. As shown in Fig. 1E, high levels of YAP/TAZ tumor formation was examined every 3 to 5 days for the duration immunoreactivity was observed in invasive tumor region. These of the experiment. For shRNA induction, xenotransplanted mice results indicate that loss of LATS1/2 is sufficient to confer potent received 2 mg/mL doxycycline in the drinking water from the first transforming activity in benign human cells. day of implantation. RNAi screening identified MK5 as a positive regulator of YAP/ Study approval TAZ The human sample experiments were reviewed and approved We aimed to find druggable targets to suppress YAP/TAZ- by the institutional review board at Severance Hospital (IRB: 4- mediated transforming activity in the context of loss of Hippo 2016-0300). The KAIST Institutional Animal Care and Use Com- pathway activity. We screened a siRNA library targeting 607 mittee approved the animal care and experimental procedures human kinases. LATS1/2-null RPE1 cells were transfected with used in this study (KA2017-26). a pool of four distinct siRNAs for each kinase for 72 hours, and YAP/TAZ immunofluorescence intensities in the nuclear and cytoplasmic (perinuclear) regions were measured (Fig. 1F). Our Results screening strategy using LATS1/2-null cells has the advantages of LATS1/2 inactivation causes constitutive YAP activation and offsetting the influence of cell density on nuclear targeting and oncogenic transformation of RPE1 cells stability of YAP/TAZ. Our image-based analysis identified MK5, Human RPE1 cells are nontransformed epithelial cells immor- MAP3K3, and PDGFRB as hits whose silencing potently decreased talized by exogenous telomerase. To establish a cell line model both nuclear levels and nuclear-to-cytoplasmic ratio of YAP/TAZ

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MK5 Inhibition Suppresses YAP-Driven Tumorigenesis

Figure 1. LATS1/2 inactivation causes constitutive YAP/TAZ activation and oncogenic transformation of RPE1 cells. A, Immunoblots of the indicated proteins in LATS1/2-wild-type and LATS1/2-null RPE1 cells. B, Immunofluorescence images showing YAP/TAZ localization of LATS1/2-wild-type and LATS1/2-null RPE1 cells. Cells were plated either at subconfluent or confluent cell density. C, Immunofluorescence micrographs visualizing YAP/TAZ and filamentous actin (phalloidin staining) in LATS1/2-null RPE1 cells treated with DMSO, cytochalasin D (Cyto D, 200 nmol/L), or blebbistatin (Blebbi, 50 mmol/L). D, Xenograft tumor growth assay of LATS1/2-wild-type and LATS1/2-null RPE1 cells in immunocompromised mice. Left, the gross and microscopic features (hematoxylin and eosin; 40) of LATS1/2-null RPE1 xenograft on day 21 after transplantation. The graph shows tumor volume changes after LATS1/2-wild-type and LATS1/2-null RPE1 xenograft. E, Immunofluorescence YAP/TAZ staining of LATS1/2-null RPE1 xenograft. F, The top diagram shows the scheme of kinome siRNA library screening. Bottom, definition of the nucleus and the cytoplasm for YAP/TAZ immunofluorescence intensity measurements using an image analysis algorithm. The nuclei of cells were identified by DAPI signals. G, The graphs show the kinome siRNA library screen results. The left graph shows nuclear YAP/TAZ staining intensities. The right graph presents the percentage of cells showing nuclear YAP/TAZ enrichment (see Materials and Methods for details). Scale bars, 15 mm(B, C, D,andF); 100 mm (E). Error bars, SEM (n ¼ 5 LATS1/2-null RPE1 xenograft and n ¼ 3 LATS1/2-wild-type xenograft in D). , P < 0.05; , P < 0.01, t test.

(Fig. 1G). Previous studies have shown that MAP3K3 interacts normalized to YAP protein levels, YAP S127 phosphorylation was with YAP (20) and PDGFRB regulates YAP activity (21). increased in parental RPE1 cells by MK5 inhibition [intensity ratio We selected MK5 for further study because knockdown of between S127-phosphorylated YAP and YAP: 1.11 (siCon) vs. MK5 caused the strongest reduction in nuclear YAP/TAZ enrich- 4.59 (siMK5); Fig. 2B]. Reduction of MYC, a potential down- ment. MK5 is a member of the atypical MAPK pathway. stream effector of YAP/TAZ (22), was also observed after MK5 Its association with the Hippo–YAP pathway is unknown. To knockdown (Fig. 2B). Moreover, YAP/TAZ target gene expression conﬁrm the screen result, we depleted MK5 in LATS1/2-null RPE1 was decreased by MK5 knockdown in LATS1/2-null RPE1 cells (Supplementary Table S2), and observed a signiﬁcant cells (Fig. 2C; Supplementary Table S3). A TEAD-responsive decrease in YAP/TAZ nuclear enrichment (Fig. 2A). In addition, luciferase assay further demonstrated decreased YAP/TAZ activity MK5 depletion reduced total levels of YAP and TAZ proteins in cells depleted of MK5 (Fig. 2D). These results indicate that in both parental and LATS1/2-null RPE1 cells (Fig. 2B). When MK5 depletion causes YAP/TAZ cytoplasmic retention and

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Figure 2. RNAi screening identified MK5 as a novel target for YAP/TAZ suppression. A, Left, immunofluorescence micrographs of LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hours. The graph is the classification and quantification of YAP/TAZ localization. Nu, mainly nuclear localization; NC, nuclear and cytoplasmic localization; Cyto, mainly cytoplasmic localization. B, Immunoblots of the indicated proteins in LATS1/2-wild-type and LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hours. C, qRT-PCR analysis of YAP/TAZ target gene expression in LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hours. D, Activity of a luciferase reporter with TEAD-binding sites in LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hours. E, Relative cell viability of LATS1/2-null RPE1 cells transfected with MK5 siRNA compared with those with control siRNA. F, Phase contrast images of LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 72 hours in 3D Matrigel culture. G, Left, Ki-67 immunofluorescence staining of LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hours. The graph shows the quantification of Ki-67–positive cells. H, Relative numbers of migrated LATS1/2-null RPE1 cells transfected with MK5 siRNA compared with those with control siRNA for 48 hours in transwell migration assay. I, Left, the immunofluorescence images of control LATS1/2-null RPE1 cells and LATS1/2-null RPE1 cells stably expressing EGFP-NES-MK5-L337A. Cells were transfected with control or MK5 siRNAs for 48 hours. The graph shows the percentage of cells exhibiting mainly nuclear YAP staining. J, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells (with/without EGFP-NES-MK5-L337A expression) transfected with control or MK5 siRNAs for 48 hours. K, qRT-PCR analysis of YAP/TAZ target genes in LATS1/2-null RPE1 cells (with/without EGFP-NES-MK5-L337A expression) transfected with control or MK5 siRNAs for 48 hours. Scale bars, 15 mm(A, G, and I); 45 mm(F). Error bars, SEM (n ¼ 3 independent experiments). , P < 0.05; , P < 0.01, t test.

degradation, leading to suppression of their transcriptional activ- suggest that MK5 inhibition suppresses the transforming activity ity in a LATS1/2-independent manner. acquired by LATS1/2 inactivation. We next tested the impact of MK5 inhibition on oncogenic transformation of LATS1/2-null RPE1 cells. MK5 knockdown MK5 promotes YAP/TAZ activity and stability decreased the viability of LATS1/2-null RPE1 cells on adherent To validate the relevance of MK5 in YAP/TAZ regulation, culture (Fig. 2E). In addition, MK5 depletion suppressed LATS1/ we tested whether exogenous siRNA-insensitive MK5 could 2-null cell proliferation in 3D Matrigel culture (Fig. 2F). We also rescue YAP/TAZ inactivation due to MK5 knockdown. MK5 is observed a decrease in the proportion of Ki-67–positive cells after knowntoremaininactiveinthenucleus, and when activated it MK5 knockdown (Fig. 2G). Decreased invasion potential of translocates to the cytoplasm (23, 24). In RPE1 cells, anti-MK5 LATS1/2-null cells after MK5 knockdown was demonstrated by immunoﬂuorescence staining was found in the nucleus, and the transwell migration assay (Fig. 2H). These results together lower levels of diffuse cytoplasmic signals were also detected

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MK5 Inhibition Suppresses YAP-Driven Tumorigenesis

(Supplementary Fig. S2A). We used an expression vector for We have reported that melanoma cell lines acquire drug constitutively active mutant MK5 tagged with nuclear exclusion resistance via YAP activation (19). Therefore, we reasoned signal and EGFP (NES-EGFP-MK5-L337A; ref. 25). We estab- that suppression of YAP activity by MK5 inhibition might lished a LATS1/2-null RPE1 cell line stably expressing the improve drug sensitivities in the resistant cancer cells. Unlike constitutively active MK5 with silent mutations in the siRNA parental SKMEL28 cells, decreased proliferation was observed target region. As shown in Fig. 2I, expression of active MK5 in PLX4032-resistant SKMEL28 cells after MK5 siRNA transfec- rescued YAP/TAZ cytoplasmic retention in LATS1/2-null RPE1 tion (Supplementary Fig. S3H). A previous study showed that cells depleted of endogenous MK5. In addition, the decrease in inhibition of cAMP-dependent protein kinase (PKA) suppresses YAP/TAZ protein levels due to MK5 knockdown was recovered MK5 activation and translocation to the cytoplasm (25). We by exogenous MK5 (Fig. 2J), and the expression of YAP/TAZ found that PKA inhibitors H89 and KT5720 reduce the protein target genes were partially rescued (Fig. 2K). Next, we tested levels of MK5, YAP, and MYC (Fig. 3F; Supplementary Fig. S3I). whetherMK5canpromoteYAP/TAZactivationinthepresence HSP27 phosphorylation that is known to be mediated by MK5 of strong inhibitory input by the Hippo pathway. We trans- was also reduced by PKA inhibitors (Fig. 3F). H89 treatment fected parental RPE1 cells with constitutively active MK5, and did not decrease the mRNA level of MK5, but the stability of treated the cells with cytochalasin D to potentiate Hippo MK5 proteins was decreased by H89 (Supplementary Fig. S3J pathway activity. Overexpression of active MK5 promoted and S3K). We tested the impact of MK5 inhibition by H89 on YAP/TAZ nuclear translocation (Supplementary Fig. S2B). A PLX4032-resistant melanoma cells. Resistant SKMEL28 and previous study showed that MK5 is activated and translocated WM3248 cells showed significant suppression of cell prolifer- to the cytoplasm by p38 MAPK agonist sodium arsenite (24). ation after combined treatment with PLX4032 and H89 Sodium arsenite treatment caused an increase in nuclear YAP/ (Fig. 3G; Supplementary Fig. S3L). Additive reduction of YAP TAZ staining as well as MK5 cytoplasmic translocation (Sup- and MYC was observed after combined treatment of PLX4032 plementary Fig. S2C). Treatment of cells with cytochalasin D and H89 (Fig. 3H; Supplementary Fig. S3M). decreased the effect of sodium arsenite on the protection of YAP To evaluate the impact of MK5 suppression on in vivo tumor- from cytoplasmic retention, which suggests competition igenesis driven by YAP overactivity, we established LATS1/2- between MK5 and the Hippo signaling. Together with the null RPE1 cells harboring doxycycline-inducible MK5 shRNA loss-of-function study using siRNA, these findings indicate that vector and confirmed doxycycline-mediated MK5 depletion MK5 is involved in positive regulation of YAP/TAZ. (Supplementary Fig. S4A). YAP/TAZ protein levels and YAP/ TAZ target gene expression were also reduced by doxycycline Inhibition of MK5 suppresses the growth of YAP-driven tumors treatment (Supplementary Fig. S4B and S4C). Notably, doxy- Malignant mesothelioma and uveal melanoma frequently have cycline induction of MK5 shRNA expression efficiently sup- mutations in NF2 (26) and GNAQ/11 (27, 28) respectively, and pressed the growth of LATS1/2-null RPE1 xenograft, as com- active YAP is important for the growth of the cancers (29). MK5 pared with control nontargeting shRNA (Fig. 3I). Without depletion decreased total YAP protein levels in the malignant doxycycline, cells containing control and MK5 shRNA showed mesothelioma cell lines MSTO-211H (carrying LATS2 mutation) similar xenograft tumor growth(SupplementaryFig.S4Dand and H2373 (carrying NF2 mutation) and the uveal melanoma cell S4E). MK5 knockdown decreased the number of mitotic cells in line 92.1 (carrying GNAQQ209L mutation; Fig. 3A). Elevated levels xenograft tumors (Fig. 3J). Decrease in Ki67-positive cells and of cleaved caspase-3, a marker of apoptosis, were observed in YAP/TAZ downregulation were also noted in the immunoflu- MK5-depleted cells, suggesting that MK5 is required for cell orescence staining of the tumors (Fig. 3K). In addition, MK5 survival (Fig. 3A). In addition, MK5 knockdown suppressed the inhibition decreased the levels of MYC, which is a downstream growth of the cancer cells in adherent and 3D Matrigel culture effector of YAP in cancers (Fig. 3L). These results consistently conditions (Fig. 3B and C). In vivo tumorigenesis capacity of show that MK5 inhibition can suppress transforming activity xenografted 211H and H2373 cells was also inhibited by MK5 and tumor growth in vivo. knockdown (Supplementary Fig. S3A). However, unlike YAP- driven cancer cells, the BRAF-driven uveal melanoma (OCM1) MK5 gene signature is associated with poor prognosis of YAP- and cutaneous melanoma (SKMEL28) cells did not show YAP/ driven cancers, and MK5 expression is correlated with YAP TAZ downregulation after MK5 depletion (Supplementary levels in human uveal melanoma specimens Fig. S3B). Viability and proliferation of OCM1 cells were also To explore transcriptome signatures of MK5 activity, we per- unaffected by MK5 depletion (Supplementary Fig. S3C and S3D). formed RNA sequencing analysis after MK5 knockdown in Activation of LATS1 by phosphorylation was detected in OCM1 LATS1/2-null RPE1 cells. Upregulated genes and downregulated cells, whereas activating phosphorylation on LATS1 was not genes upon MK5 depletion were identified [P < 0.05 and absolute detected in 92.1 cells (Supplementary Fig. S3E). These results log2 (fold change) 2; Fig. 4A]. A gene ontology analysis revealed suggest that YAP regulation by MK5 acts when YAP is active and that cell migration, cell cycle, and cell proliferation were most that MK5 is a suitable target for inhibiting YAP activity in YAP- enriched functional categories of the downregulated genes driven cancers. We next targeted MK5 in 92.1 cells using CRISPR- (Fig. 4B). Enriched functional categories of the upregulated genes Cas9 system. MK5-null 92.1 cells were generated (Supplementary upon MK5 depletion are shown in Supplementary Fig. S5A. As Fig. S3F), and showed YAP and CTGF downregulation (Fig. 3D; expected, gene-set enrichment analysis (GSEA) showed enrich- Supplementary Fig. S3G). In MK5-null 92.1 cells, overexpression ment of YAP signature genes upon MK5 depletion (Fig. 4C). The of MK5 increased YAP protein levels (Fig. 3E). Additional trans- expression of RB, MTOR, E2F1 signature genes were also influ- fection of YAP siRNA decreased the level of MYC (Fig. 3E), enced by MK5 (Fig. 4C). To validate the clinical significance of suggesting that MK5 changes the expression of MYC through YAP MK5 in human cancers, correlations between MK5 and YAP regulation. signatures and the impact of MK5 gene signature on patient

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Figure 3. Inhibition of MK5 suppresses the growth of YAP-driven tumors. A, Immunoblots of the indicated proteins in malignant mesothelioma (MSTO-211H and H2373) and uveal melanoma (92.1) cells transfected with control or MK5 siRNAs for 72 hours. B, Relative cell viability of YAP-driven malignant mesothelioma and uveal melanoma cells transfected with MK5 siRNA compared with those with control siRNA for 72 and 96 hours. C, Phase contrast images of YAP-driven malignant mesothelioma and uveal melanoma cells in 3D Matrigel culture. Cells were transfected with control or MK5 siRNA for 72 hours and further incubated for 48 hours. D, Immunoblots of the indicated proteins in MK5-wild-type and MK5-null 92.1 cells. E, Immunoblots of the indicated proteins in MK5-null 92.1 cells transfected with the indicated siRNAs and plasmids for 72 hours. F, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with DMSO or H89 (10 mmol/L) for 6 hours. G, Cell viability assay of PLX4032-resistant SKMEL28 cells treated with PLX4032 (PLX, 2 mmol/L), H89 (10 mmol/L), or their combinations for 72 hours. H, Immunoblots of the indicated proteins in PLX4032-resistant SKMEL28 cells treated with PLX4032 (2 mmol/L), H89 (10 mmol/L), or their combinations for 24 hours. I, Xenograft tumor growth assay of LATS1/2-null RPE1 cells expressing control or doxycycline-inducible MK5 shRNA. Xenotransplanted mice received 2 mg/mL doxycycline in the drinking water from the first day of implantation and tumors were harvested 29 days after transplantation. Left, the gross features of LATS1/2-null RPE1 xenograft. The graph shows tumor growth. J, Left, the hematoxylin and eosin staining of a xenograft tumor obtained from the experiment presented in I. Arrows, mitotic cells. The graph is the quantification of mitotic cells at 10 high power fields (HPF) by hematoxylin and eosin staining. K, Immunofluorescence images of YAP/TAZ and Ki-67 of a xenograft tumor obtained from the experiment presented in I. L, Immunofluorescence staining of MYC of a xenograft tumor obtained from the experiment presented in I. Scale bars, 75 mm(C); 1 mm (I); 30 mm(J–L). Error bars, SEM (n ¼ 3 independent experiments; n ¼ 4 mice). , P < 0.05; , P < 0.01, t test.

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Figure 4. MK5 is a potential therapeutic target for YAP-driven cancers. A, Left, the MA-plot comparing MK5 versus control siRNA knockdown in LATS1/2-null RPE1 cells.

Significantly upregulated genes (blue) and downregulated genes (red) were highlighted using cutoff of P < 0.05 and absolute log2 (fold change) 2. Right, heatmap showing significantly downregulated and upregulated genes by MK5 siRNA knockdown in LATS1/2-null RPE1 cells. B, The graph shows significantly enriched gene ontology terms in 343 downregulated genes upon MK5 knockdown. The number of genes and –log P value in the gene ontology terms are shown. C, GSEA plots showing significantly enriched gene signatures in LATS1/2-null RPE1 cells after control siRNA knockdown versus those after MK5 siRNA knockdown. D, The scatterplot shows correlation between MK5 and YAP gene signatures in TCGA database. Right, Kaplan–Meier curves comparing overall survival of patients with MK5 gene signature high versus low in TCGA database. E, Scatterplots and survival curves for different cancer types from the analysis presented in D. Mesothelioma, TCGA-MESO; lung adenocarcinoma, TCGA-LUAD; lower grade glioma, TCGA-LGG. F, Representative figures of YAP and MK5 IHC staining in human uveal melanoma (UVM) tissues. Scale bar, 5 mm. The tables show the expression of MK5 and the activity of YAP in 80 primary and 16 metastatic uveal melanoma samples.

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survival were evaluated by analyzing The Cancer Genome Atlas interacting proteins, including LATS1/2. Because MK5 contains (TCGA) data. We defined the MK5 gene signature as 343 down- PPFY sequence at residues 239–242, we tested whether the WW regulated genes upon MK5 knockdown and used it with YAP domain of YAP is essential for MK5 binding. Unexpectedly, GSEA gene signature (YAP1_UP, Broad Institute) for a correlation deletion of the WW domain resulted in a slight decrease in YAP analysis. Analysis using all 11,023 TCGA cases across 33 cancer binding to MK5 (Fig. 5J). In contrast, deletion of the transcription types showed that the MK5 gene signature correlated with YAP activation domain (TAD) abolished YAP binding to MK5 gene signature as well as poor survival of patients (Fig. 4D). (Fig. 5K). TAD deletion did not interfere with LATS1 binding of Cancer types presenting high MK5 gene signature are shown in YAP (Fig. 5K). YAP contains a phosphodegron sequence in the Supplementary Fig. S5B. Notably, cancer types showing strong middle of the TAD domain. As shown in Fig. 5L, deletion of the correlation for MK5 and YAP signatures exhibited poor prognosis phosphodegron sequence remarkably weakened YAP binding to according to high MK5 gene signature (Fig. 4E). MK5. This suggests that the phosphodegron region is not essential Next, to confirm MK5-mediated YAP regulation in YAP- but is important for the strong interaction between YAP and MK5. driven cancers, we investigated the association between YAP In accordance with the absence or reduction of binding activity, and MK5 expression in uveal melanoma tissue specimens. IHC the protein levels of TAD- or phosphodegron-deleted YAP was staining of YAP and MK5 was performed in specimens from 96 not increased by coexpression of MK5 (Supplementary Fig. S6B). UVM samples (80 primary and 16 metastatic uveal melanoma). Although not statistically significant, MK5 knockdown Notably, tumors with higher levels of MK5 expression showed decreased YAP mRNA levels (Supplementary Fig. S6C). Treatment higher YAP activity or expression. YAP nuclear localization of cells with verteporfin, which inhibits YAP protein activity, also was positively correlated with higher MK5 expression in both reduced YAP mRNA levels (Supplementary Fig. S6C). Thus, it is patients with primary uveal melanoma and metastatic uveal possible that YAP activity is directly or indirectly involved in the melanoma (Fig. 4F). The level of YAP IHC staining intensity transcription of the YAP gene. The cycloheximide chase assay was also correlated with MK5 expression levels (Supplementary showed that the half-life of YAP proteins increased in LATS1/2- Fig. S5C). Upregulation of MYC was observed in tumors with null cells overexpressing MK5, as compared with that in control high MK5 expression at a marginal significance (P ¼ 0.082; cells (Fig. 6A). Reduction of mRNA levels may contribute to YAP Supplementary Fig. S5D). There was no significant difference downregulation by MK5 knockdown. However, because protein in the expression of MK5 or YAP between primary and meta- level regulation appeared to be a more direct mode of MK5 action, static tumors (Supplementary Fig. S5D). Taken together, these we focused on the regulation of YAP protein stability by MK5. The results suggest that YAP expression is associated with MK5 ubiquitin–proteasome pathway and the autolysosomal pathway expression in human cancers, and that MK5-YAP is a potential are two major protein degradation pathways in mammalian therapeutic target. cells (30). We investigated the relevance of these pathways in YAP stability regulation by MK5. Inhibition of autophagic flux by MK5 physically interacts with YAP and protects proteasomal bafilomycin A did not influence MK5 siRNA–mediated YAP degradation of YAP degradation (Fig. 6B). In contrast, blocking proteasome activity We next explored the mechanism of YAP regulation by MK5. by MG132 rescued YAP/TAZ downregulation by MK5 knock- We examined potential physical interactions between YAP and down. The stability of exogenously expressed YAP was also MK5. Reciprocal coimmunoprecipitation indicated that exoge- increased by proteasome inhibition (Fig. 6C). To confirm that nously expressed YAP and MK5 are in the same protein complex MK5 regulates YAP stability via the ubiquitin–proteasome path- (Fig. 5A and B). Interactions between endogenous YAP and MK5 way, we performed an ubiquitination assay using HA-tagged proteins in LATS1/2-null RPE1 cells and 92.1 cells were also ubiquitin. Stably expressed YAP was immunoprecipitated and demonstrated by immunoprecipitation analyses (Fig. 5C and the effect of MK5 overexpression on YAP ubiquitination was D). We examined the distribution of overexpressed exogenous analyzed by Western blotting. MK5 overexpression decreased the YAP and MK5 in RPE1 cells. Exogenous YAP and MK5 showed ubiquitination of YAP (Fig. 6D). Taken together, these data clear colocalization in the perinuclear cytoplasm and the cell suggest that MK5 increases YAP stability by preventing proteaso- periphery (Fig. 5E). Along with their coimmunoprecipitation, mal degradation of YAP. this result suggests that MK5 physically interacts with YAP to influence YAP stability and activity. To further investigate MK5– MK5–YAP interaction counteracts CK1d/e-mediated YAP YAP interaction, we generated a series of C-terminal MK5 deletion degradation constructs (Fig. 5F) and coexpressed the deletion mutants with Casein kinase 1d/e (CK1d/e) acts as a regulator of YAP stability YAP. MK5 lacking NES/NLS and c-terminal residues coprecipi- in collaboration with the core Hippo kinases. Phosphorylation 1-200 tated with YAP, but MK5 did not precipitate YAP, suggesting in the phosphodegron of YAP by CK1d/e induces ubiquitination that MK5 residues 201–322 are required for the interaction and proteasomal degradation (31). Because LATS1/2-mediated with YAP (Fig. 5G and H). Consistently, MK5 residues 1–200 YAP-Ser381 phosphorylation is a key trigger for YAP phosphor- did not colocalize with exogenous YAP, whereas MK5 lacking ylation by CK1d/e (31), we speculated that CK1d/e would not NES/NLS and c-terminal residues colocalized with YAP (Supple- affect YAP abundance in the absence of LATS1/2. However, the mentary Fig. S6A). We observed an increase in exogenous YAP level of YAP protein was increased by CK1d/e inhibition in protein levels when coexpressed with full-length MK5 (Fig. 5G). LATS1/2-null background (Fig. 6E and F; Supplementary The C-terminal deletion mutants that bind to YAP did not Fig. S6D). Notably, the decrease in YAP protein levels caused increased YAP levels, suggesting that C-terminal residues are by MK5 depletion was rescued by CK1d/e and MK5 coknock- required for increasing YAP stability. down (Fig. 6G). To validate the counteraction between MK5 We next generated YAP deletion constructs (Fig. 5I). The WW and CK1d/e in YAP regulation, we overexpressed MK5 and CK1e domains of YAP are known to bind to the PPxY motif of its in LATS1/2-null RPE1 cells. Consistent with the knockdown

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Figure 5. MK5 physically interacts with YAP and protects proteasomal degradation of YAP. A, Coimmunoprecipitation analysis of YAP and MK5 with lysates from RPE1 cells stably expressing FLAG-YAP vector. IP, immunoprecipitation; IB, immunoblot. B, Coimmunoprecipitation analysis of MK5 and YAP with lysates from LATS1/ 2-null RPE1 cells stably expressing V5-MK5 vector. C and D, Immunoprecipitation of endogenous YAP and MK5 in LATS1/2-null RPE1 and 92.1 cells. E, Immunoﬂuorescence images showing colocalization of exogenous YAP and MK5 in RPE1 cells. Bottom, magniﬁed view of the boxed area. Scale bar, 5 mm. F, Schematic representations of the C-terminal MK5 deletion constructs. G and H, Coimmunoprecipitation analysis of YAP and the indicated MK5 deletion constructs with lysates from RPE1 cells stably expressing FLAG-YAP vector. I, Schematic representations of the YAP deletion constructs. J–L, Coimmunoprecipitation analysis of MK5 and the indicated YAP deletion constructs with lysates from RPE1 cells stably expressing V5-MK5.

experiment result, exogenous CK1e decreased the level of YAP ical interaction between MK5 and CK1e was detected by immu- protein, and MK5 overexpression rescued the YAP downregula- noprecipitation (Supplementary Fig. S6E). Exogenous MK5 tion (Fig. 6H). Moreover, MK5 reversed the enhanced ﬂag-YAP and CK1e showed colocalization in the cell periphery as well ubiquitination caused by CK1e overexpression (Fig. 6I). Phys- as in the cytoplasm of LATS1/2-null RPE1 cells (Supplementary

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Figure 6. MK5–YAP interaction counteracts CK1d/e-mediated YAP degradation. A, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells. Cells were transfected with empty or V5-MK5 vector for 24 hours and treated with cycloheximide (CHX, 100 mg/mL). Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells (B) and RPE1 cells stably expressing FLAG-YAP vector (C). Cells were transfected with control or MK5 siRNAs for 36 hours and further treated with baﬁlomycin A (Baf A, 10 nmol/L) or MG132 (10 mmol/L) for 6 hours. D, Ubiquitination analysis of precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with HA-ubiquitin (HA-Ub) and V5-MK5 expression vectors. Cells were treated with MG132 (10 mmol/L) for 6 hours after 24 hours of transfection. IP,

immunoprecipitation; IB, immunoblot. E, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells transfected with control or CK1d/e siRNAs for 72 hours. F, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with PF670462 (100 nmol/L, 500 mmol/L, or 1 mmol/L) for 24 hours. G, Immunoblots

of the indicated proteins in LATS1/2-null RPE1 cells transfected with control, MK5, or MK5 and CK1d/e siRNAs for 48 hours. H, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells transfected with empty, CK1e, or CK1e and MK5 vectors for 24 hours. I, Ubiquitination analysis of precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with the indicated plasmids for 24 hours and treated with MG132 (10 mmol/L) for 6 hours. J, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells transfected with control, MK5, or MK5 and b-TRCP siRNAs for 48 hours. K, Immunoprecipitation assay of endogenous b-TRCP with precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with empty or V5-MK5 vector.

Fig. 6F), suggesting that YAP, MK5, and CK1e together are MK5 kinase activity is required for YAP protection recruitedtoaspeciﬁc scaffold structure. Phosphorylated YAP The kinase activity of MK5 is required for the stability and phosphodegron recruits the b-TRCP subunit of the SCF E3 proper subcellular localization of MK5 (24). K51E mutation in ubiquitin ligase complex, which leads to YAP degradation (31). the ATP-binding domain is known to block MK5 kinase activi- As expected, the decrease in YAP protein levels due to ty (24). Transiently expressed wild-type MK5 localized to the MK5 depletion was rescued by b-TRCP and MK5 coknockdown cytoplasm (Supplementary Fig. S7A). MK5 may be activated by (Fig. 6J). Coimmunoprecipitation analysis showed that MK5 liposome-mediated transfection stress. In contrast, transiently inhibited the physical interaction between YAP and b-TRCP expressed kinase-dead MK5 mainly localized to the nucleus (Fig. 6K). Taken together, these results indicate that MK5 (Supplementary Fig. S7A). Although intact MK5 and YAP exhib- counteracts CK1d/e-mediated YAP ubiquitination. ited clear colocalization in the cytoplasm, kinase-dead MK5 did

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Figure 7. MK5 kinase activity is required for YAP protection. A, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells. Cells were transfected with empty, wild- type, or kinase-dead (K51E) MK5 vector for 24 hours. B, Ubiquitination analysis of precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with the indicated plasmids for 24 hours and treated with MG132 (10 mmol/L) for 3 hours. C, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with DMSO or MK2/MK3/MK5 inhibitor (PF3644022, 50 mmol/L) for 48 hours. D, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with DMSO, PF3644022 (50 mmol/L, 48 hours), or PF3644022 (50 mmol/L, 48 hours), and PF670462 (1 mmol/L, 12 hours). E, Coimmunoprecipitation analysis with lysates from RPE1 cells stably expressing FLAG-YAP vector. Cells were transfected with V5-MK5 vector and incubated in medium with DMSO or PF3644022 (50 mmol/L) for 24 hours. F, Immunoblots of the indicated proteins in RPE1 cells stably expressing FLAG-YAP-5SA transfected with control or MK5 siRNAs for 48 hours. G, Immunoﬂuorescence images of RPE1 cells stably expressing FLAG-YAP-5SA transfected with control or MK5 siRNAs for 48 hours. H, Coimmunoprecipitation analysis of the MK5 mutants with precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transiently transfected with EGFP-NES-MK5 or EGFP-NES-MK5-K51E vector. I, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells. Cells were transiently transfected with the indicated MK5 vectors. J, A schematic model. Scale bar, 12 mm(G).

not cause relocalization of cytoplasmic YAP into the nucleus (Fig. 7B). Exogenously expressed YAP was decreased when (Supplementary Fig. S7B). This suggests that physical interaction YAP–MK5 interaction was weakened by H89 treatment or between kinase-dead MK5 and YAP is not strong enough to retain kinase-dead MK5 overexpression (Supplementary Fig. S7C). YAP in the nucleus. Kinase-dead MK5 showed little effect on total PF3644022 is a reversible ATP-competitive compound that inhi- YAP protein levels (Fig. 7A), indicating a critical role for MK5 bits the kinase activity of MK2 and MK5 with high selectivity and kinase activity in YAP protection. In addition, overexpression of potency (32). The close family member MK3 shows 10-fold kinase-dead MK5 failed to protect YAP from ubiquitination weaker response to PF3644022 (32). Treatment of cells with

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PF3644022 decreased the level of YAP and MYC as well as models promotes tumorigenesis, and genetic or epigenetic silenc- phosphor-HSP27 (Fig. 7C). The reduction of YAP by PF3644022 ing of Hippo component genes LATS1/2, FAT1, and NF2 have treatment was rescued by combined treatment with PF3644022 been identified in various cancer types (10, 26, 37). Therefore, and the CK1d/e inhibitor PF670482 (Fig. 7D). Notably, the Hippo-off or Hippo-low contexts are likely to be the source of YAP binding activity between YAP and MK5 was inhibited by overactivity during the progression of YAP-driven tumorigenesis. PF3644022 (Fig. 7E). Unlike MK5, knockdown of MK2 did not Rigorous investigations on Hippo-independent YAP regulation change YAP levels, and MK3 knockdown increased YAP (Supple- will be critical for finding optimal therapeutic strategies for YAP- mentary Fig. S7D). Thus, the effect of PF3644022 on YAP down- driven cancers. b regulation appears to be due to MK5 inhibition. The mechanism Several ubiquitin E3 ligases, including SCF -TRCP, are known to of YAP upregulation by MK3 inhibition requires further investi- be involved in the regulation of YAP protein stability (38). gation. Taken together, these results suggest that selective inhi- Deregulation of LATS1 protein stability regulators, such as bition of MK5 kinase activity have therapeutic potential to sup- NEDD4 E3 ligases, contributes to tumor-selective YAP overex- press YAP function. pression (39). Another study demonstrated that FBXW7 is a major Although overexpression of wild-type MK5 increased YAP E3 ubiquitin ligase for YAP degradation, loss of which promotes abundance, phosphorylation patterns of YAP were not noticeably YAP overexpression in HCC cells (40). Our results indicate that changed by MK5 in both LATS1/2 wild-type and LATS1/2-null MK5 positively regulates YAP abundance, counteracting YAP cells (Supplementary Fig. S7E and S7F). MK5 overexpression also ubiquitination and proteasomal degradation (Figs. 6 and 7). did not significantly alter the phosphorylation status of CK1e Reducing YAP protein stability by MK5 inhibition enables sup- (Supplementary Fig. S7G and S7H). The level of YAP-5SA, which pression of tumor growth in xenograft model (Fig. 3), which harbors mutations in five LATS phosphorylation motifs, was confirms the potential of YAP stability modulation as a targeted decreased by MK5 knockdown (Fig. 7F and G). These results therapy for YAP-driven cancers. Previous studies have shown that suggest that regulation of YAP phosphorylation may not be the the degradation of YAP is primed during sequential YAP phos- main mechanism of YAP protection by MK5. We next tested phorylation by LATS1/2 and CK1d/e (31). However, we found whether a cytoplasmic form of kinase-dead MK5 can interact that CK1d/e still possess the capacity to induce YAP degradation in with and protect YAP. Cytoplasmic forms of kinase-active and LATS1/2-null cells, suggesting that other LATS1/2-like kinases kinase-dead MK5 were coprecipitated with YAP at a similar level perform the priming phosphorylation and affect YAP abundance (Fig. 7H). In addition, cytoplasmic form of kinase-dead MK5 in the absence of LATS1/2. We propose that physical interaction increased the level of coexpressed YAP (Fig. 7I), suggesting that between YAP/MK5 and MK5/CK1e in the cytoplasm may interfere MK5 kinase activity in YAP protection is required for cytoplasmic with the priming YAP phosphorylation and/or subsequent acti- translocation of MK5. vation of YAP phosphodegron by CK1d/e. Depletion of HSP27, the downstream component of YAP, did MK5 was originally identified as a phosphorylation target of the not clearly affect the level of YAP in LATS1/2-null RPE1 cells p38 MAP kinase (24). Later studies have demonstrated that MK5 (Supplementary Fig. S8A). Depletion of ERK3 and ERK4, major activation is also mediated by the atypical MAPKs ERK3/4 upstream regulators of MK5, also did not affect YAP (Supple- (33, 34, 41). In the downstream of MK5, HSP27, p53, Foxo1, mentary Fig. S8B and S8C; refs. 33, 34). Therefore, YAP stability and Rheb are potential phosphorylation substrates by MK5 (42). regulation may be unrelated to the previously recognized MK5 MK5 is highly conserved throughout the vertebrates, and known signaling pathway. MK5 knockdown did not consistently affect to be involved in diverse cellular processes, including cell metab- the expression and activation of core Hippo kinases (Supplemen- olism (43), autophagy (44), senescence (45), and actin cytoskel- tary Fig. S8D). Conditions that decrease LATS activity did not eton rearrangement (25). However, the specific extracellular changed MK5 localization (Supplementary Fig. S8E). stimuli controlling the activity of MK5 has not yet been fully We propose a YAP stability regulation model in which MK5 established and biological functions of MK5 remain unclear in binding to YAP in the cytoplasm counteracts CK1d/e-mediated many cellular activities. The knockout mouse studies targeting YAP ubiquitination and degradation (Fig. 7J). MK5 kinase activity MK5 exon 8 indicated a tumor suppressive role of MK5 in skin is required for YAP regulation, presumably because of its role in papilloma development after a chemical carcinogen treat- MK5 cytoplasmic translocation. It is not clear how MK5 is ment (45), but later studies argued both the reliability of MK5 involved in YAP nuclear translocation in the LATS1/2-null knockout method and the tumor-suppressive effect of MK5 background. knockout (46). Our study newly incorporates MK5 in the Hip- po–YAP pathway regulation, raising the possibility that MK5 involves various cellular processes via regulation of YAP activity. Discussion Investigating MK5–YAP axis may expand our understanding of Previous RNAi and compound library screens for modulators MK5-related biologic processes. of the Hippo–YAP pathway were performed in cells with intact MK5 inhibition may exhibit its antitumor activity in YAP- Hippo activity, and revealed multiple upstream inputs regulating driven cancers by cell-cycle suppression and MYC downregula- the Hippo kinases (8, 35, 36). In contrast, we used LATS1/2-null tion. Previous studies showed E2F1-related cell-cycle progres- cells to discover a positive regulator of YAP that can be targeted in sion (47) and MYC activation (22) as core downstream effectors YAP-driven cancers. The complex inputs to the Hippo kinase of YAP-driven tumorigenesis. Although MYC was shown to be cascade are blocked in LATS1/2-null background. The use of negatively regulated by MK5 in some cellular contexts (48), MK5 LATS1/2-null cells in the screen contributed to avoid the identi- depletion induced MYC downregulation as well as E2F target gene fication of false or indirect hits due to the confounding factors. In downregulation (Figs. 2 and 4). Our RNA-seq analysis estab- addition, Hippo-independent YAP regulators have relevance to lished the MK5-related gene signature, majorly of which are YAP-driven cancers. Deletion of Hippo components in mouse enriched with YAP target genes and cell-cycle–related genes.

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Remarkably, MK5-related gene signature correlates with the YAP Disclosure of Potential Conflicts of Interest gene signature as well as poor survival of the patients (Fig. 4). We No potential conflicts of interest were disclosed. expect that MK5 inhibition has a great therapeutic potential not only in suppressing YAP-driven tumorigenesis, but in inhibiting Authors' Contributions other YAP-driven cancer pathogenesis, including resistance to Conception and design: J. Seo, M.H. Kim, J. Kim molecular targeted agents in melanoma (19). Development of methodology: J. Seo, M.H. Kim, H. Hong Several kinases that control YAP activity have been identified as Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Seo, M.H. Kim, S.K. Kim mediators of the Hippo-independent mechanisms in YAP regu- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, lation. The cellular energy sensor AMPK phosphorylates YAP and computational analysis): J. Seo, M.H. Kim, H. Hong, S. Park, S.K. Kim, H. Cho thus inhibits the YAP–TEAD interaction (15). In addition, YAP Writing, review, and/or revision of the manuscript: J. Seo, M.H. Kim, S.K. Kim, phosphorylation by AMPK or the virus-activated kinase IKKe J. Kim triggers lysosomal degradation of YAP (49). AKT phosphorylates Administrative, technical, or material support (i.e., reporting or organizing YAP at Ser127, resulting in cytoplasmic retention of YAP (50). The data, constructing databases): J. Seo, M.H. Kim, H. Hong Study supervision: J. Kim c-ABL tyrosine kinase and the Src family tyrosine kinases, such as SRC, YES, and LCK, phosphorylate YAP at Tyr357 (51–53). Although YAP Tyr357 phosphorylation increases YAP stability Acknowledgments and nuclear enrichment, the phosphorylation inhibits YAP- We thank Ugo Moens (The University of Tromsø, Tromsø, Norway) for – providing EGFP-NES-MK5 plasmid and Hyun Woo Park (Yonsei University, TEAD induced transcription and allows YAP to interact with Seoul, South Korea) for providing uveal melanoma and malignant meso- other transcription factors. For example, DNA damage–induced thelioma cell lines. We thank Dae-Sik Lim (KAIST) for providing SBP-FLAG- phosphorylation of YAP by c-ABL promotes the binding of YAP YAP plasmid and its deletion derivatives. We also appreciate Won Do Heo and p73 to induce the expression of proapoptotic target (KAIST) for providing high-throughput imaging system (ImageXpress) and genes (52). Because all of these kinases negatively regulate onco- Young Seok Ju (KAIST) for providing comments on bioinformatic analysis. fi genic YAP-TEAD transcriptional activity or promote YAP degra- We acknowledge board-certi ed pathologist Seokhwi Kim (KAIST) for interpreting the pathology findings and mitotic figures of LATS1/2-null dation, the inhibition of these kinases is not a candidate for an RPE1 xenografts. We thank June-Koo Lee (Harvard Medical School, Boston, anticancer strategy. In contrast, MK5 positively regulates both YAP MA) for instructing the CRISPR/Cas9-based gene editing method. This stability and YAP-TEAD transcriptional activity. Thus, MK5 is a research was supported by Basic Science Research Program through the suitable target for suppressing YAP overactivity with kinase National Research Foundation of Korea funded by the Korean Ministry of inhibitors. Science and ICT (2017R1A2B3005208). In summary, we propose a new strategy that targets YAP stability in YAP-driven cancers where Hippo signaling is inacti- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked vated by genetic or epigenetic alterations. MK5, which protects advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate YAP degradation in a Hippo-independent manner, would be a this fact. potential therapeutic target for YAP-driven cancers. Our findings prompt the need for further investigation of YAP degradation Received April 30, 2019; revised August 21, 2019; accepted September 27, pathways in cancer. 2019; published first October 2, 2019.

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6152 Cancer Res; 79(24) December 15, 2019 Cancer Research

MK5 Regulates YAP Stability and Is a Molecular Target in YAP-Driven Cancers

Jimyung Seo, Min Hwan Kim, Hyowon Hong, et al.

Cancer Res 2019;79:6139-6152. Published OnlineFirst October 2, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-1339

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