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

Author Manuscript Published OnlineFirst on October 2, 2019; DOI: 10.1158/0008-5472.CAN-19-1339 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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#, Joon Kim1#

1Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea. 2Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea. 3Department of Pathology, Yonsei University College of Medicine, Seoul, Korea. *These authors contributed equally to this work. #Co-corresponding authors

Running title: MK5 inhibition suppresses YAP-driven tumorigenesis

Corresponding authors: Joon Kim, PhD Associate Professor Graduate School of Medical Science and Engineering Korea Advanced Institute of Science and Technology (KAIST) 291 Daehak-ro, Daejeon 34141, Republic of Korea Phone: +82-42-350-4242 E-mail: [email protected]

Sang Kyum Kim, MD/PhD Assistant Professor Department of Pathology Yonsei University College of Medicine 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea Phone: +82-1599-1004 E-mail: [email protected]

Disclosure of Conflicts of interest: No potential conflicts of interest were disclosed.

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 2, 2019; DOI: 10.1158/0008-5472.CAN-19-1339 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Transcriptional regulator YAP is activated in multiple human cancers and plays critical roles in tumor initiation, progression, metastasis, and drug resistance. However, therapeutic targeting of the Hippo-YAP pathway has been challenging due to its low druggability and limited knowledge of YAP regulation in cancer. Here we present a functional screen and identify a novel therapeutic target for YAP-driven tumorigenesis. RNAi screening using an oncogenic YAP activation model identified the serine/threonine kinase MK5 as a positive regulator of YAP. MK5 physically interacted with YAP and counteracted CK1δ/ε-mediated YAP ubiquitination and degradation independent of LATS1/2. MK5 kinase activity was essential for protecting YAP from ubiquitin- mediated degradation and cytoplasmic retention. Downregulating MK5 expression inhibited the survival of YAP-activated cancer cell lines and mouse xenograft models. MK5 upregulation was associated with high levels of YAP expression and poor prognosis in clinical tumor samples, confirming its important role for YAP activity in human cancer. These results uncover MK5 as a novel factor that regulates YAP stability and that targeting the YAP degradation pathway controlled by MK5 is a potential strategy for suppressing YAP activity in cancer.

Significance

Findings reveal MK5 is a novel kinase that regulates YAP in a LATS-independent manner and can be targeted for cancer therapy.

Introduction

Cancer genome sequencing efforts have uncovered several signaling pathways in which major components are frequently mutated in human cancers (1). Although robust clinical activity of EGFR, ALK, and BRAF-targeted drugs has been demonstrated, it is lagging behind to find new effective targets beyond the receptor tyrosine kinase pathway for anticancer drug development. Recent studies highlight the association of the Hippo-YAP pathway with principal cancer features (2, 3). Overactivation of YAP can induce tumorigenesis, and YAP is also associated with cancer stem cell properties, epithelial mesenchymal transition, metastasis, and drug resistance (3). Targeting the Hippo-YAP pathway is expected to be beneficial not only for suppressing tumorigenesis and tumor growth, but also for overcoming metastasis and resistance to anticancer agents (2, 3). However, despite intense recent interest, clinical targeting of the Hippo-YAP pathway has been challenging due to low druggability of the pathway and limited understanding of its regulatory mechanisms in human cancer. In the canonical Hippo-YAP pathway, sequential phosphorylation of the core Hippo kinases MST and LATS leads to phosphorylation of the pathway effector YAP and its paralog TAZ (3). Phosphorylation by LATS1/2 inhibits the transcriptional activity of YAP through cytoplasmic sequestration and proteasomal degradation. The core Hippo kinases and their adaptors are regarded as tumor suppressors (4). Previous studies uncovered a number of regulatory inputs for the Hippo kinases, including GPCRs (5), Rho GTPases (6), the actin cytoskeleton (7), the mevalonate pathway (8), and hypoxia (9). However, the Hippo signaling pathway does not have a designated ligand-receptor pair, making it difficult to develop a specific agonist for the pathway. Considering that Hippo kinase activity is affected by multiple cellular cues, targeting one of the regulatory inputs would result in modest anti-tumor activity. Moreover, the expression of the Hippo kinases is frequently blocked by genetic alterations (10, 11). Direct inhibition of the binding between YAP and its partner transcription factor TEAD is also challenging because inhibition of the protein-protein interaction is difficult due to flat and large interfaces lacking targetable binding pockets (12). Verteporfin, a YAP antagonist known to interfere with YAP-TEAD complex formation, has low YAP inhibitory efficacy (13) and low specificity protein crosslinking activity (14). The hurdles in targeting the Hippo signaling

pathway or YAP-TEAD interaction make it attractive to target non-canonical YAP regulatory mechanisms that are active in YAP-driven cancers. A number of studies have demonstrated that the activity and stability of YAP can be regulated by cellular metabolic status (15), osmotic stress (16), and autophagy (17) in a Hippo-independent manner. These Hippo-independent YAP regulatory factors play a complex role in various cellular processes, and thus pharmacological manipulation of these factors will induce pleiotropic effects. Therefore, there is a need for unbiased screening approach to identify novel regulators of YAP activity or stability that are good drug targets. In the present study, we performed cell-based RNA interference (RNAi) library screening to identify genes required for nuclear enrichment of YAP. Our screening identified the serine/threonine kinase MK5 (also called MAPKAPK5 or PRAK) as a novel positive regulator of YAP. We found that ubiquitination and degradation of YAP is protected by MK5. Moreover, we provide evidence that MK5 is a useful target molecule for developing drugs for YAP-driven cancers including uveal melanoma (UVM) and malignant mesothelioma (MM).

Materials and Methods

Cell culture Cells were acquired from ATCC and large frozen stocks were made to ensure against contaminations by other cell lines. All cell lines were used within 10 passages after reviving from the frozen stocks. Cells were free of Mycoplasma contamination as determined by staining cells with DAPI every 2 or 3 passages. Cells were cultured in the following media: [RPE1 cells: DMEM/F12 (Welgene); MSTO-211H, H2373, 92.1, and OCM1 cells: RPMI1640 (Welgene); SKMEL cells: MEM (Welgene); WM3248 cells: MCDB 153 (Welgene)] supplemented with 10 % FBS and 1 % penicillin/streptomycin. RPE1 cell lines carrying inactivating mutations in both LATS1 and LATS2 genes were established using the CRISPR-Cas9 system according to the previously published protocol (18). PLX4032-resistant SKMEL28 and WM3248 cells were established according to the previously published protocol (19).

Kinome siRNA library screening A kinome-wide siRNA library targeting 607 human kinases (Dharmacon) was used in this study. Two biologic replicates of LATS1/2 knockout RPE1 cells were transfected with target siRNAs, fixed, and stained. Subcellular YAP/TAZ immunofluorescence was analyzed by CellProfiler software (Broad Institute). Detailed methods are presented in the supplementary material.

Xenograft For xenograft experiments, female nude mice (6 weeks old) were injected subcutaneously with the indicated cells and the tumor formation was examined every 3 to 5 days for the duration of the experiment. For shRNA induction, xenotransplanted mice received 2 mg/ml doxycycline in the drinking water from the first day of implantation.

Study approval The human sample experiments were reviewed and approved by the institutional review board at Severance Hospital (IRB: 4-2016-0300). The KAIST IACUC approved the animal care and experimental procedures used in this study (KA2017-26).

Results

LATS1/2 inactivation causes constitutive YAP activation and oncogenic transformation of RPE1 cells Human RPE1 cells are non-transformed epithelial cells immortalized by exogenous telomerase. To establish a cell line model that mimics the context in which the Hippo signaling pathway is inactivated, we edited LATS1 and LATS2 in RPE1 cells using the CRISPR-Cas9 system (Supplementary Fig. S1A). Frameshift mutations in both copies of LATS1 (exon 5) and LATS2 (exon 3) were introduced, and the loss of LATS1/2 expression was confirmed by an immunoblot assay (Fig. 1A and Supplementary Table S1). As expected, YAP phosphorylation was substantially reduced by LATS1/2 inactivation (Fig. 1A and Supplementary Fig. S1B). Unlike parental RPE1 cells, LATS1/2-null RPE1 cells exhibited nuclear YAP/TAZ enrichment even at high cell densities (Fig. 1B). YAP/TAZ nuclear localization in LATS1/2-null RPE1 cells was not suppressed by inhibitors of actin polymerization or actomyosin contraction (Fig. 1C). Transient co-transfection of LATS1 and LATS2 caused YAP/TAZ cytoplasmic retention, confirming that constitutive YAP/TAZ nuclear localization is attributed to the inactivation of LATS1/2 (Supplementary Fig. S1C). Residual YAP phosphorylation at serine 127 (S127) was not reduced by LATS1/2 siRNA transfection (Supplementary Fig. S1D). Along with YAP/TAZ nuclear localization, the expression of YAP/TAZ target genes was upregulated in LATS1/2-null RPE1 cells (Supplementary Fig. S1E). Consistent with the fact that the Hippo pathway mediates contact inhibition of cell proliferation, LATS1/2-null cells continued to proliferate at high cell densities (Supplementary Fig. S1F). The rate of cell proliferation and the fraction of S/G2/M phase cells were also increased in LATS1/2-null context (Supplementary Fig. S1G and H). We next examined whether LATS1/2 loss resulted in oncogenic transformation of RPE1 cells. Parental RPE1 cells failed to grow in 3-dimensional (3D) matrigel culture, whereas LATS1/2-null RPE1 cells were highly proliferative in matrigel (Supplementary Fig. S1I). Nuclear localization of YAP/TAZ in LATS1/2-null RPE1 cells persisted in matrigel culture (Supplementary Fig. S1J). A transwell migration assay showed that LATS1/2 loss significantly increased invasion capacity of RPE1 cells (Supplementary Fig. S1K). Moreover, in vivo tumorigenic potential of LATS1/2-null RPE1

cells was demonstrated by xenograft into immunocompromised mice (BALB/c nude). LATS1/2- null RPE1 cells efficiently formed tumors after transplantation, whereas parental RPE1 cells did not develop detectable tumors (Fig. 1D). Haematoxylin and eosin staining of the LATS1/2-null RPE1 tumor showed pleomorphism and increased nuclear to cytoplasm ratio, consistent with typical findings of carcinoma. As shown in Fig. 1E, high levels of YAP/TAZ immunoreactivity was observed in invasive tumor region. These results indicate that loss of LATS1/2 is sufficient to confer potent transforming activity in benign human cells.

RNAi screening identified MK5 as a positive regulator of YAP/TAZ We aimed to find druggable targets to suppress YAP/TAZ-mediated transforming activity in the context of loss of Hippo pathway activity. We screened a siRNA library targeting 607 human kinases. LATS1/2-null RPE1 cells were transfected with a pool of four distinct siRNAs for each kinase for 72 hr, and YAP/TAZ immunofluorescence intensities in the nuclear and cytoplasmic (perinuclear) regions were measured (Fig. 1F). Our screening strategy using LATS1/2-null cells has the advantages of offsetting the influence of cell density on nuclear targeting and stability of YAP/TAZ. Our image-based analysis identified MK5, MAP3K3, and PDGFRB as hits whose silencing potently decreased both nuclear levels and nucleocytoplasmic ratio of YAP/TAZ (Fig. 1G). Previous studies have shown that MAP3K3 interacts with YAP (20) and PDGFRB regulates YAP activity (21). We selected MK5 for further study because knockdown of MK5 caused the strongest reduction in nuclear YAP/TAZ enrichment. MK5 is a member of the atypical MAP kinase (MAPK) pathway. Its association with the Hippo-YAP pathway is unknown. To confirm the screen result, we depleted MK5 in LATS1/2-null RPE1 cells (Supplementary Table S2), and observed a significant decrease in YAP/TAZ nuclear enrichment (Fig. 2A). In addition, MK5 depletion reduced total levels of YAP and TAZ proteins in both parental and LATS1/2-null RPE1 cells (Fig. 2B). When normalized to YAP protein levels, YAP S127 phosphorylation was increased in parental RPE1 cells by MK5 inhibition [intensity ratio between S127- phosphorylated YAP and YAP: 1.11 (siCon) vs 4.59 (siMK5); Fig. 2B]. Reduction of MYC, a potential downstream effector of YAP/TAZ (22), was also observed after MK5 knockdown (Fig. 2B). Moreover, YAP/TAZ target gene expression was decreased by MK5 knockdown in LATS1/2-null RPE1 cells (Fig. 2C and Supplementary Table S3). A TEAD-responsive luciferase assay further demonstrated decreased YAP/TAZ activity in cells depleted of MK5 (Fig. 2D).

These results indicate that MK5 depletion causes YAP/TAZ cytoplasmic retention and degradation, leading to suppression of their transcriptional activity in a LATS1/2-independent manner. We next tested the impact of MK5 inhibition on oncogenic transformation of LATS1/2-null RPE1 cells. MK5 knockdown decreased the viability of LATS1/2-null RPE1 cells on adherent culture (Fig. 2E). In addition, MK5 depletion suppressed LATS1/2-null cell proliferation in 3D matrigel culture (Fig. 2F). We also observed a decrease in the proportion of Ki-67 positive cells after MK5 knockdown (Fig. 2G). Decreased invasion potential of LATS1/2-null cells after MK5 knockdown was demonstrated by the transwell migration assay (Fig. 2H). These results together suggest that MK5 inhibition suppresses the transforming activity acquired by LATS1/2 inactivation.

MK5 promotes YAP/TAZ activity and stability To validate the relevance of MK5 in YAP/TAZ regulation, we tested whether exogenous siRNA- insensitive MK5 could rescue YAP/TAZ inactivation due to MK5 knockdown. MK5 is known to remain inactive in the nucleus, and when activated it translocates to the cytoplasm (23, 24). In RPE1 cells, anti-MK5 immunofluorescnece staining was found in the nucleus, and lower levels of diffuse cytoplasmic signals were also detected (Supplementary Fig. S2A). We used an expression vector for constitutively active mutant MK5 tagged with nuclear exclusion signal and EGFP (NES-EGFP-MK5-L337A) (25). We established a LATS1/2-null RPE1 cell line stably expressing the constitutively active MK5 with silent mutations in the siRNA target region. As shown in Fig. 2I, expression of active MK5 rescued YAP/TAZ cytoplasmic retention in LATS1/2-null RPE1 cells depleted of endogenous MK5. In addition, the decrease in YAP/TAZ protein levels due to MK5 knockdown was recovered by exogenous MK5 (Fig. 2J), and the expression of YAP/TAZ target genes were partially rescued (Fig. 2K). Next, we tested whether MK5 can promote YAP/TAZ activation in the presence of strong inhibitory input by the Hippo pathway. We transfected parental RPE1 cells with constitutively active MK5, and treated the cells with cytochalasin D to potentiate Hippo pathway activity. Overexpression of active MK5 promoted YAP/TAZ nuclear translocation (Supplementary Fig. S2B). A previous study showed that MK5 is activated and translocated to the cytoplasm by p38 MAPK agonist sodium arsenite (24). Sodium arsenite treatment caused an increase in nuclear YAP/TAZ staining as well as MK5

cytoplasmic translocation (Supplementary Fig. S2C). Treatment of cells with cytochalasin D decreased the effect of sodium arsenite on the protection of YAP from cytoplasmic retention, which suggests competition between MK5 and the Hippo signaling. Together with the loss-of- function study using siRNA, these findings indicate that MK5 is involved in positive regulation of YAP/TAZ.

Inhibition of MK5 suppresses the grow th of YAP-driven tumors MM and UVM frequently have mutations in NF2 (26) and GNAQ/11 (27, 28) respectively, and active YAP is important for the growth of the cancers (29). MK5 depletion decreased total YAP protein levels in the MM cell lines MSTO-211H (carrying LATS2 mutation) and H2373 (carrying NF2 mutation) and the UVM cell line 92.1 (carrying GNAQQ209L mutation) (Fig. 3A). Elevated levels of cleaved caspase 3, a marker of apoptosis were observed in MK5-depleted cells, suggesting that MK5 is required for cell survival (Fig. 3A). In addition, MK5 knockdown suppressed the growth of the cancer cells in adherent and 3D matrigel culture conditions (Fig. 3B and C). In vivo tumorigenesis capacity of xenografted 211H and H2373 cells was also inhibited by MK5 knockdown (Supplementary Fig. S3A). However, unlike YAP-driven cancer cells, the BRAF-driven UVM (OCM1) and cutaneous melanoma (SKMEL28) cells did not show YAP/TAZ downregulation after MK5 depletion (Supplementary Fig. S3B). Viability and proliferation of OCM1 cells were also unaffected by MK5 depletion (Supplementary Fig. S3C and D). Activation of LATS1 by phosphorylation was detected in OCM1 cells, whereas activating phosphorylation on LATS1 was not detected in 92.1 cells (Supplementary Fig. S3E). These results suggest that YAP regulation by MK5 acts when YAP is active and that MK5 is a suitable target for inhibiting YAP activity in YAP-driven cancers. We next targeted MK5 in 92.1 cells using CRISPR-Cas9 system. MK5-null 92.1 cells were generated (Supplementary Fig. S3F), and showed YAP and CTGF downregulation (Fig. 3D and Supplementary Fig. S3G). In MK5- null 92.1 cells, overexpression of MK5 increased YAP protein levels (Fig. 3E). Additional transfection of YAP siRNA decreased the level of MYC (Fig. 3E), suggesting that MK5 changes the expression of MYC through YAP regulation. We have reported that melanoma cell lines acquire drug resistance via YAP activation (19). Therefore, we reasoned that suppression of YAP activity by MK5 inhibition might improve drug sensitivities in the resistant cancer cells. Unlike parental SKMEL28 cells, decreased proliferation

was observed in PLX4032-resistant SKMEL28 cells after MK5 siRNA transfection (Supplementary Fig. S3H). A previous study showed that inhibition of cAMP-dependent protein kinase (PKA) suppresses MK5 activation and translocation to the cytoplasm (25). We found that PKA inhibitors H89 and KT5720 reduce the protein levels of MK5, YAP, and MYC (Fig. 3F and Supplementary Fig. S3I). HSP27 phosphorylation that is known to be mediated by MK5 was also reduced by PKA inhibitors (Fig. 3F). H89 treatment did not decrease the mRNA level of MK5, but the stability of MK5 proteins was decreased by H89 (Supplementary Fig. S3J and K). We tested the impact of MK5 inhibition by H89 on PLX4032-resistant melanoma cells. Resistant SKMEL28 and WM3248 cells showed significant suppression of cell proliferation after combined treatment with PLX4032 and H89 (Fig. 3G and Supplementary Fig. S3L). Additive reduction of YAP and MYC was observed after combined treatment of PLX4032 and H89 (Fig. 3H and Supplementary Fig. S3M). To evaluate the impact of MK5 suppression on in vivo tumorigenesis driven by YAP overactivity, we established LATS1/2-null RPE1 cells harboring doxycycline-inducible MK5 shRNA vector and confirmed doxycycline-mediated MK5 depletion (Supplementary Fig. S4A). YAP/TAZ protein levels and YAP/TAZ target gene expression were also reduced by doxycycline treatment (Supplementary Fig. S4B and C). Notably, doxycycline induction of MK5 shRNA expression efficiently suppressed the growth of LATS1/2-null RPE1 xenograft, as compared to control non-targeting shRNA (Fig. 3I). Without doxycycline, cells containing control and MK5 shRNA showed similar xenograft tumor growth (Supplementary Fig. S4D and E). MK5 knockdown decreased the number of mitotic cells in xenograft tumors (Fig. 3J). Decrease in Ki67-positive cells and YAP/TAZ downregulation were also noted in the immunofluorescence staining of the tumors (Fig. 3K). In addition, MK5 inhibition decreased the levels of MYC, which is a downstream effector of YAP in cancers (Fig. 3L). These results consistently show that MK5 inhibition can suppress transforming activity and tumor growth in vivo.

MK5 gene signature is associated with poor prognosis of YAP-driven cancers, and MK5 expression is correlated with YAP levels in human UVM specimens To explore transcriptome signatures of MK5 activity, we performed RNA sequencing analysis after MK5 knockdown in LATS1/2-null RPE1 cells. Upregulated genes and downregulated genes upon MK5 depletion were identified (p-value < 0.05 and absolute log2 (fold change) ≥ 2) (Fig.

4A). A gene ontology analysis revealed that cell migration, cell cycle, and cell proliferation were most enriched functional categories of the downregulated genes (Fig. 4B). Enriched functional categories of the upregulated genes upon MK5 depletion are shown in Supplementary Fig. S5A. As expected, gene set enrichment analysis (GSEA) showed enrichment of YAP signature genes upon MK5 depletion (Fig. 4C). The expression of RB, MTOR, E2F1 signature genes were also influenced by MK5 (Fig. 4C). To validate the clinical significance of MK5 in human cancers, correlations between MK5 and YAP signatures and the impact of MK5 gene signature on patient survival were evaluated by analyzing TCGA data. We defined the MK5 gene signature as 343 downregulated genes upon MK5 knockdown and used it with YAP GSEA gene signature (YAP1_UP, Broad Institute) for a correlation analysis. Analysis using all 11,023 TCGA cases across 33 cancer types showed that the MK5 gene signature correlated with YAP gene signature as well as poor survival of patients (Fig. 4D). Cancer types presenting high MK5 gene signature are shown in Supplementary Fig. S5B. Notably, cancer types showing strong correlation for MK5 and YAP signatures exhibited poor prognosis according to high MK5 gene signature (Fig 4E). Next, to confirm MK5-mediated YAP regulation in YAP-driven cancers, we investigated the association between YAP and MK5 expression in UVM tissue specimens. Immunohistochemical (IHC) staining of YAP and MK5 was performed in specimens from 96 UVM samples (80 primary and 16 metastatic UVM). Notably, tumors with higher levels of MK5 expression showed higher YAP activity or expression. YAP nuclear localization was positively correlated with higher MK5 expression in both primary UVM and metastatic UVM patients (Fig. 4F). The level of YAP IHC staining intensity was also correlated with MK5 expression levels (Supplementary Fig. S5C). Upregulation of MYC was observed in tumors with high MK5 expression at a marginal significance (p = 0.082; Supplementary Fig. S5D). There was no significant difference in the expression of MK5 or YAP between primary and metastatic tumors (Supplementary Fig. S5D). Taken together, these results suggest that YAP expression is associated with MK5 expression in human cancers, and that MK5-YAP is a potential therapeutic target.

MK5 physically interacts with YAP and protects proteasomal degradation of YAP We next explored the mechanism of YAP regulation by MK5. We examined potential physical interactions between YAP and MK5. Reciprocal co-immunoprecipitation indicated that

exogenously expressed YAP and MK5 are in the same protein complex (Fig. 5A and B). Interactions between endogenous YAP and MK5 proteins in LATS1/2-null RPE1 cells and 92.1 cells were also demonstrated by immunoprecipitation analyses (Fig. 5C and D). We examined the distribution of overexpressed exogenous YAP and MK5 in RPE1 cells. Exogenous YAP and MK5 showed clear co-localization in the perinuclear cytoplasm and the cell periphery (Fig. 5E). Along with their co-immunoprecipitation, this result suggests that MK5 physically interacts with YAP to influence YAP stability and activity. To further investigate MK5-YAP interaction, we generated a series of C-terminal MK5 deletion constructs (Fig. 5F) and co-expressed the deletion mutants with YAP. MK5 lacking NES/NLS and c-terminal residues co-precipitated with YAP, but MK51-200 did not precipitate YAP, suggesting that MK5 residues 201-322 are required for the interaction with YAP (Fig. 5G and H). Consistently, MK5 residues 1-200 did not co-localize with exogenous YAP, whereas MK5 lacking NES/NLS and c-terminal residues co-localized with YAP (Supplementary Fig. S6A). We observed an increase in exogenous YAP protein levels when co- expressed with full-length MK5 (Fig. 5G). The C-terminal deletion mutants that bind to YAP did not increased YAP levels, suggesting that C-terminal residues are required for increasing YAP stability. We next generated YAP deletion constructs (Fig. 5I). The WW domains of YAP are known to bind to the PPxY motif of its interacting proteins, including LATS1/2. Because MK5 contains PPFY sequence at residues 239-242, we tested whether the WW domain of YAP is essential for MK5 binding. Unexpectedly, deletion of the WW domain resulted in a slight decrease in YAP binding to MK5 (Fig. 5J). In contrast, deletion of the transcription activation domain (TAD) abolished YAP binding to MK5 (Fig. 5K). TAD deletion did not interfere with LATS1 binding of YAP (Fig. 5K). YAP contains a phosphodegron sequence in the middle of the TAD domain. As shown in Fig. 5L, deletion of the phosphodegron sequence remarkably weakened YAP binding to MK5. This suggests that the phosphodegron region is not essential but is important for the strong interaction between YAP and MK5. In accordance with the absence or reduction of binding activity, the protein levels of TAD- or phosphodegron-deleted YAP was not increased by co- expression of MK5 (Supplementary Fig. S6B). Although not statistically significant, MK5 knockdown decreased YAP mRNA levels (Supplementary Fig. S6C). Treatment of cells with verteporfin, which inhibits YAP protein activity, also reduced YAP mRNA levels (Supplementary Fig. S6C). Thus, it is possible that YAP

activity is directly or indirectly involved in the transcription of the YAP gene. The cycloheximide chase assay showed that the half-life of YAP proteins increased in LATS1/2-null cells overexpressing MK5, as compared with that in control cells (Fig. 6A). Reduction of mRNA levels may contribute to YAP downregulation by MK5 knockdown. However, because protein level regulation appeared to be a more direct mode of MK5 action, we focused on the regulation of YAP protein stability by MK5. The ubiquitin-proteasome pathway and the autolysosomal pathway are two major protein degradation pathways in mammalian cells (30). We investigated the relevance of these pathways in YAP stability regulation by MK5. Inhibition of autophagic flux by bafilomycin A did not influence MK5 siRNA-mediated YAP degradation (Fig. 6B). In contrast, blocking proteasome activity by MG132 rescued YAP/TAZ downregulation by MK5 knockdown. The stability of exogenously expressed YAP was also increased by proteasome inhibition (Fig. 6C). To confirm that MK5 regulates YAP stability via the ubiquitin-proteasome pathway, we performed an ubiquitination assay using HA-tagged ubiquitin. Stably expressed YAP was immunoprecipitated and the effect of MK5 overexpression on YAP ubiquitination was analyzed by western blotting. MK5 overexpression decreased the ubiquitination of YAP (Fig. 6D). Taken together, these data suggest that MK5 increases YAP stability by preventing proteasomal degradation of YAP.

MK5-YAP interaction counteracts CK1δ/ε-mediated YAP degradation

Casein kinase 1δ/ε (CK1δ/ε) acts as a regulator of YAP stability in collaboration with the core

Hippo kinases. Phosphorylation in the phosphodegron of YAP by CK1δ/ε induces ubiquitination and proteasomal degradation (31). Because LATS1/2-mediated YAP-Ser381 phosphorylation is a

key trigger for YAP phosphorylation by CK1δ/ε (31), we speculated that CK1δ/ε would not affect YAP abundance in the absence of LATS1/2. However, the level of YAP protein was increased by

CK1δ/ε inhibition in LATS1/2-null background (Fig. 6E and F; Supplementary Fig. S6D).

Notably, the decrease in YAP protein levels caused by MK5 depletion was rescued by CK1δ/ε and

MK5 co-knockdown (Fig. 6G). To validate the counteraction between MK5 and CK1δ/ε in YAP

regulation, we overexpressed MK5 and CK1ε in LATS1/2-null RPE1 cells. Consistent with the

knockdown experiment result, exogenous CK1ε decreased the level of YAP protein, and MK5 overexpression rescued the YAP downregulation (Fig. 6H). Moreover, MK5 reversed the

enhanced flag-YAP ubiquitination caused by CK1ε overexpression (Fig. 6I). Physical interaction

between MK5 and CK1ε was detected by immunoprecipitation (Supplementary Fig. S6E).

Exogenous MK5 and CK1ε showed co-localization in the cell periphery as well as in the cytoplasm of LAT S 1 /2 -null RPE1 cells (Supplementary Fig. 6F), suggesting that YAP, MK5 and

CK1ε together are recruited to a specific scaffold structure. Phosphorylated YAP phosphodegron recruits the β-TRCP subunit of the SCF E3 ubiquitin ligase complex, which leads to YAP degradation (31). As expected, the decrease in YAP protein levels due to MK5 depletion was rescued by β-TRCP and MK5 co-knockdown (Fig. 6J). Co-immunoprecipitation analysis showed that MK5 inhibited the physical interaction between YAP and β-TRCP (Fig. 6K). Taken together,

these results indicate that MK5 counteracts CK1δ/ε-mediated YAP ubiquitination.

MK5 kinase activity is required for YAP protection The kinase activity of MK5 is required for the stability and proper subcellular localization of MK5 (24). K51E mutation in the ATP-binding domain is known to block MK5 kinase activity (24). Transiently expressed wild type MK5 localized to the cytoplasm (Supplementary Fig. S7A). MK5 may be activated by liposome-mediated transfection stress. In contrast, transiently expressed kinase-dead MK5 mainly localized to the nucleus (Supplementary Fig. S7A). Although intact MK5 and YAP exhibited clear colocalization in the cytoplasm, kinase-dead MK5 did not cause relocalization of cytoplasmic YAP into the nucleus (Supplementary Fig. S7B). This suggests that physical interaction between kinase-dead MK5 and YAP is not strong enough to retain YAP in the nucleus. Kinase-dead MK5 showed little effect on total YAP protein levels (Fig. 7A), indicating a critical role for MK5 kinase activity in YAP protection. In addition, overexpression of kinase-dead MK5 failed to protect YAP from ubiquitination (Fig. 7B). Exogenously expressed YAP was decreased when YAP-MK5 interaction was weakened by H89 treatment or kinase-dead MK5 overexpression (Supplementary Fig. S7C). PF3644022 is a reversible ATP-competitive compound that inhibits the kinase activity of MK2 and MK5 with high selectivity and potency (32). The close family member MK3 shows 10-fold weaker response to PF3644022 (32). Treatment of cells with PF3644022 decreased the level of YAP and MYC as well as phosphor-HSP27 (Fig. 7C). The reduction of YAP by PF3644022 treatment was

rescued by combined treatment with PF3644022 and the CK1δ/ε inhibitor PF670482 (Fig. 7D). Notably, the binding activity between YAP and MK5 was inhibited by PF3644022 (Fig. 7E).

Unlike MK5, knockdown of MK2 did not change YAP levels, and MK3 knockdown increased YAP (Supplementary Fig. S7D). Thus, the effect of PF3644022 on YAP downregulation appears to be due to MK5 inhibition. The mechanism of YAP upregulation by MK3 inhibition requires further investigation. Taken together, these results suggest that selective inhibition of MK5 kinase activity have therapeutic potential to suppress YAP function. Although overexpression of wild type MK5 increased YAP abundance, phosphorylation patterns of YAP were not noticeably changed by MK5 in both LATS1/2 wild type and LATS1/2 null cells (Supplementary Fig. S7E and F). MK5 overexpression also did not significantly alter the phosphorylation status of CK1ε (Supplementary Fig. S7G and H). The level of YAP-5SA, which harbors mutations in five LATS phosphorylation motifs, was decreased by MK5 knockdown (Fig. 7F and G). These results suggest that regulation of YAP phosphorylation may not be the main mechanism of YAP protection by MK5. We next tested whether a cytoplasmic form of kinase-dead MK5 can interact with and protect YAP. Cytoplasmic forms of kinase-active and kinase-dead MK5 were co-precipitated with YAP at a similar level (Fig. 7H). In addition, cytoplasmic form of kinase-dead MK5 increased the level of co-expressed YAP (Fig. 7I), suggesting that MK5 kinase activity in YAP protection is required for cytoplasmic translocation of MK5. Depletion of HSP27, the downstream component of YAP, did not clearly affect the level of YAP in LATS1/2-null RPE1 cells (Supplementary Fig. S8A). Depletion of ERK3 and ERK4, major upstream regulators of MK5, also did not affect YAP (Supplementary Fig. S8B and C) (33, 34). Therefore, YAP stability regulation may be unrelated to the previously recognized MK5 signaling pathway. MK5 knockdown did not consistently affect the expression and activation of core Hippo kinases (Supplementary Fig. S8D). Conditions that decrease LATS activity did not changed MK5 localization (Supplementary Fig. S8E). We propose a YAP stability regulation model in which MK5 binding to YAP in the cytoplasm counteracts CK1δ/ε-mediated YAP ubiquitination and degradation (Fig. 7J). MK5 kinase activity is required for YAP regulation, presumably because of its role in MK5 cytoplasmic translocation. It is not clear how MK5 is involved in YAP nuclear translocation in the LATS1/2-null background.

Discussion

Previous RNAi and compound library screens for modulators of the Hippo-YAP pathway were performed in cells with intact Hippo activity, and revealed multiple upstream inputs regulating the Hippo kinases (8, 35, 36). In contrast, we used LATS1/2-null cells to discover a positive regulator of YAP that can be targeted in YAP-driven cancers. The complex inputs to the Hippo kinase cascade are blocked in LATS1/2-null background. The use of LATS1/2-null cells in the screen contributed to avoid the identification of false or indirect hits due to the confounding factors. In addition, Hippo-independent YAP regulators have relevance to YAP-driven cancers. Deletion of Hippo components in mouse models promotes tumorigenesis, and genetic or epigenetic silencing of Hippo component genes LATS1/2, FAT1, and NF2 have been identified in various cancer types (10, 26, 37). Therefore, Hippo-off or Hippo-low contexts are likely to be the source of YAP overactivity during the progression of YAP-driven tumorigenesis. Rigorous investigations on Hippo-independent YAP regulation will be critical for finding optimal therapeutic strategies for YAP-driven cancers. Several ubiquitin E3 ligases, including SCFβ-TRCP, are known to be involved in the regulation of YAP protein stability (38). Deregulation of LATS1 protein stability regulators, such as NEDD4 E3 ligases, contributes to tumor-selective YAP overexpression (39). Another study demonstrated that FBXW7 is a major E3 ubiquitin ligase for YAP degradation, loss of which promotes YAP overexpression in HCC cells (40). Our results indicate that MK5 positively regulates YAP abundance, counteracting YAP ubiquitination and proteasomal degradation (Fig. 6 and 7). Reducing YAP protein stability by MK5 inhibition enables suppression of tumor growth in xenograft model (Fig. 3), which confirms the potential of YAP stability modulation as a targeted therapy for YAP-driven cancers. Previous studies have shown that the degradation of YAP is primed during sequential YAP phosphorylation by LATS1/2 and CK1δ/ε (31). However,

we found that CK1δ/ε still possess the capacity to induce YAP degradation in LATS1/2-null cells, suggesting that other LATS1/2-like kinases perform the priming phosphorylation and affect YAP abundance in the absence of LATS1/2. We propose that physical interaction between YAP/MK5

and MK5/CK1ε in the cytoplasm may interfere with the priming YAP phosphorylation and/or

subsequent activation of YAP phosphodegron by CK1δ/ε.

MK5 was originally identified as a phosphorylation target of the p38 MAP kinase (24). Later studies have demonstrated that MK5 activation is also mediated by the atypical MAP kinases ERK3/4 (33, 34, 41). In the downstream of MK5, HSP27, p53, Foxo1, and Rheb are potential phosphorylation substrates by MK5 (42). MK5 is highly conserved throughout the vertebrates, and known to be involved in diverse cellular processes, including cell metabolism (43), autophagy (44), senescence (45), and actin cytoskeleton rearrangement (25). However, the specific extracellular stimuli controlling the activity of MK5 has not yet been fully established and biological functions of MK5 remain unclear in many cellular activities. The knockout mouse studies targeting MK5 exon 8 indicated a tumor suppressive role of MK5 in skin papilloma development after a chemical carcinogen treatment (45), but later studies argued both the reliability of MK5 knockout method and the tumor suppressive effect of MK5 knockout (46). Our study newly incorporates MK5 in the Hippo-YAP pathway regulation, raising the possibility that MK5 involves various cellular processes via regulation of YAP activity. Investigating MK5- YAP axis may expand our understanding of MK5-related biologic processes. MK5 inhibition may exhibit its antitumor activity in YAP-driven cancers by cell cycle suppression and MYC downregulation. Previous studies showed E2F1-related cell cycle progression (47) and MYC activation (22) as core downstream effectors of YAP-driven tumorigenesis. Although MYC was shown to be negatively regulated by MK5 in some cellular contexts (48), MK5 depletion induced MYC downregulation as well as E2F target gene downregulation (Fig. 2 and 4). Our RNA-seq analysis established the MK5-related gene signature, majorly of which are enriched with YAP target genes and cell cycle related genes. Remarkably, MK5-related gene signature correlates with the YAP gene signature as well as poor survival of the patients (Fig. 4). We expect that MK5 inhibition has a great therapeutic potential not only in suppressing YAP-driven tumorigenesis, but in inhibiting other YAP-driven cancer pathogenesis, including resistance to molecular targeted agents in melanoma (19). Several kinases that control YAP activity have been identified as mediators of the Hippo- independent mechanisms in YAP regulation. The cellular energy sensor AMPK phosphorylates YAP and thus inhibits the YAP-TEAD interaction (15). In addition, YAP phosphorylation by AMPK or the virus-activated kinase IKKε triggers lysosomal degradation of YAP (49). AKT phosphorylates YAP at Ser127, resulting in cytoplasmic retention of YAP (50). The 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 and nuclear enrichment, the phosphorylation inhibits YAP-TEAD-induced transcription and allows YAP to interact with other transcription factors. For example, DNA damage-induced phosphorylation of YAP by c-ABL promotes the binding of YAP and p73 to induce the expression of proapoptotic target genes (52). Because all of these kinases negatively regulate oncogenic YAP-TEAD transcriptional activity or promote YAP degradation, the inhibition of these kinases is not a candidate for an anticancer strategy. In contrast, MK5 positively regulates both YAP stability and YAP-TEAD transcriptional activity. Thus, MK5 is a suitable target for suppressing YAP overactivity with kinase inhibitors. In summary, we propose a new strategy that targets YAP stability in YAP-driven cancers where Hippo signaling is inactivated by genetic or epigenetic alterations. MK5, which protects YAP degradation in a Hippo-independent manner, would be a potential therapeutic target for YAP-driven cancers. Our findings prompt the need for further investigation of YAP degradation pathways in cancer.

Acknowledgments

We thank Ugo Moens (The University of Tromsø) for providing EGFP-NES-MK5 plasmid and Hyun Woo Park (Yonsei University) for providing uveal melanoma and malignant mesothelioma cell lines. We thank Dae-Sik Lim (KAIST) for providing SBP-FLAG-YAP plasmid and its deletion derivatives. We also appreciate Won Do Heo (KAIST) for providing high throughput imaging system (ImageXpress), and Young Seok Ju (KAIST) for providing comments on bioinformatic analysis. We acknowledge board certified pathologist Seokhwi Kim (KAIST) for interpreting the pathology findings and mitotic figures of LATS1/2-null RPE1 xenografts. We thank June-Koo Lee (Harvard Medical School) for instructing the CRISPR/Cas9-based gene editing method. This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Korean Ministry of Science and ICT (2017R1A2B3005208).

References

1. Sanchez-Vega F, Mina M, Armenia J, Chatila WK, Luna A, La KC, et al. Oncogenic Signaling Pathways in The Cancer Genome Atlas. Cell 2018;173:321-37. 2. Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ at the Roots of Cancer. Cancer Cell 2016;29:783- 803. 3. Yu FX, Zhao B, Guan KL. Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer. Cell 2015;163:811-28. 4. Koontz LM, Liu-Chittenden Y, Yin F, Zheng Y, Yu J, Huang B, et al. The Hippo effector Yorkie controls normal tissue growth by antagonizing scalloped-mediated default repression. Dev Cell 2013;25:388-401. 5. Yu FX, Zhao B, Panupinthu N, Jewell JL, Lian I, Wang LH, et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 2012;150:780-91. 6. Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, et al. Role of YAP/TAZ in mechanotransduction. Nature 2011;474:179-83. 7. Aragona M, Panciera T, Manfrin A, Giulitti S, Michielin F, Elvassore N, et al. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 2013;154:1047-59. 8. Sorrentino G, Ruggeri N, Specchia V, Cordenonsi M, Mano M, Dupont S, et al. Metabolic control of YAP and TAZ by the mevalonate pathway. Nat Cell Biol 2014;16:357-66. 9. Ma B, Chen Y, Chen L, Cheng H, Mu C, Li J, et al. Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase. Nat Cell Biol 2015;17:95-103. 10. Yu T, Bachman J, Lai ZC. Mutation analysis of large tumor suppressor genes LATS1 and LATS2 supports a tumor suppressor role in human cancer. Protein Cell 2015;6:6-11. 11. Mehra R, Vats P, Cieslik M, Cao X, Su F, Shukla S, et al. Biallelic Alteration and Dysregulation of the Hippo Pathway in Mucinous Tubular and Spindle Cell Carcinoma of the Kidney. Cancer Discov 2016;6:1258-66. 12. Thangudu RR, Bryant SH, Panchenko AR, Madej T. Modulating protein-protein interactions with small molecules: the importance of binding hotspots. J Mol Biol 2012;415:443-53. 13. Warren JSA, Xiao Y, Lamar JM. YAP/TAZ Activation as a Target for Treating Metastatic Cancer. Cancers (Basel) 2018;10(4). 14. Konstantinou EK, Notomi S, Kosmidou C, Brodowska K, Al-Moujahed A, Nicolaou F, et al. Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity. Sci Rep 2017;7:46581. 15. Mo JS, Meng Z, Kim YC, Park HW, Hansen CG, Kim S, et al. Cellular energy stress induces AMPK- mediated regulation of YAP and the Hippo pathway. Nat Cell Biol 2015;17:500-10. 16. Lin KC, Moroishi T, Meng Z, Jeong HS, Plouffe SW, Sekido Y, et al. Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation. Nat Cell Biol 2017;19:996-1002. 17. Liang N, Zhang C, Dill P, Panasyuk G, Pion D, Koka V, et al. Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex. J Exp Med 2014;211:2249-63. 18. Park HW, Kim YC, Yu B, Moroishi T, Mo JS, Plouffe SW, et al. Alternative Wnt Signaling Activates YAP/TAZ. Cell 2015;162:780-94. 19. Kim MH, Kim J, Hong H, Lee SH, Lee JK, Jung E, et al. Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation. EMBO J 2016;35:462-78.

20. Chen TH, Chen CY, Wen HC, Chang CC, Wang HD, Chuu CP, et al. YAP promotes myogenic differentiation via the MEK5-ERK5 pathway. FASEB J 2017;31:2963-72. 21. Smoot RL, Werneburg NW, Sugihara T, Hernandez MC, Yang L, Mehner C, et al. Platelet-derived growth factor regulates YAP transcriptional activity via Src family kinase dependent tyrosine phosphorylation. J Cell Biochem 2018;119:824-36. 22. Choi W, Kim J, Park J, Lee DH, Hwang D, Kim JH, et al. YAP/TAZ Initiates Gastric Tumorigenesis via Upregulation of MYC. Cancer Res 2018;78:3306-20. 23. Deleris P, Rousseau J, Coulombe P, Rodier G, Tanguay PL, Meloche S. Activation loop phosphorylation of the atypical MAP kinases ERK3 and ERK4 is required for binding, activation and cytoplasmic relocalization of MK5. J Cell Physiol 2008;217:778-88. 24. Seternes OM, Johansen B, Hegge B, Johannessen M, Keyse SM, Moens U. Both binding and activation of p38 mitogen-activated protein kinase (MAPK) play essential roles in regulation of the nucleocytoplasmic distribution of MAPK-activated protein kinase 5 by cellular stress. Mol Cell Biol 2002;22:6931-45. 25. Gerits N, Mikalsen T, Kostenko S, Shiryaev A, Johannessen M, Moens U. Modulation of F-actin rearrangement by the cyclic AMP/cAMP-dependent protein kinase (PKA) pathway is mediated by MAPK-activated protein kinase 5 and requires PKA-induced nuclear export of MK5. J Biol Chem 2007;282:37232-43. 26. Sekido Y. Inactivation of Merlin in malignant mesothelioma cells and the Hippo signaling cascade dysregulation. Pathol Int 2011;61:331-44. 27. Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O'Brien JM, et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009;457:599-602. 28. Van Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S, Wiesner T, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010;363:2191-9. 29. Feng X, Degese MS, Iglesias-Bartolome R, Vaque JP, Molinolo AA, Rodrigues M, et al. Hippo- independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry. Cancer Cell 2014;25:831-45. 30. Nam T, Han JH, Devkota S, Lee HW. Emerging Paradigm of Crosstalk between Autophagy and the Ubiquitin-Proteasome System. Mol Cells 2017;40:897-905. 31. Zhao B, Li L, Tumaneng K, Wang CY, Guan KL. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP). Genes Dev 2010;24:72-85. 32. Mourey R, Burnette B, Brustkern S, Daniels JS, Hirsch J, Hood W, et al. A Benzothiophene Inhibitor of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Inhibits Tumor Necrosis Factor α Production and Has Oral Anti-Inflammatory Efficacy in Acute and Chronic Models of Inflammation. J Pharmacol Exp Ther 2010;333:797-807. 33. Kant S, Schumacher S, Singh MK, Kispert A, Kotlyarov A, Gaestel M. Characterization of the atypical MAPK ERK4 and its activation of the MAPK-activated protein kinase MK5. J Biol Chem 2006;281:35511-9. 34. Aberg E, Perander M, Johansen B, Julien C, Meloche S, Keyse SM, et al. Regulation of MAPK- activated protein kinase 5 activity and subcellular localization by the atypical MAPK ERK4/MAPK4. J Biol Chem 2006;281:35499-510. 35. Mohseni M, Sun J, Lau A, Curtis S, Goldsmith J, Fox VL, et al. A genetic screen identifies an LKB1- MARK signalling axis controlling the Hippo-YAP pathway. Nat Cell Biol 2014;16:108-17. 36. Pascual-Vargas P, Cooper S, Sero J, Bousgouni V, Arias-Garcia M, Bakal C. RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer. Sci Data

2017;4:170018. 37. Martin D, Degese MS, Vitale-Cross L, Iglesias-Bartolome R, Valera JLC, Wang Z, et al. Assembly and activation of the Hippo signalome by FAT1 tumor suppressor. Nat Commun 2018;9:2372. 38. Nguyen TH, Kugler JM. Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer. Cancers (Basel) 2018;10(4). 39. Salah Z, Cohen S, Itzhaki E, Aqeilan RI. NEDD4 E3 ligase inhibits the activity of the Hippo pathway by targeting LATS1 for degradation. Cell cycle 2013;12:3817-23. 40. Tu K, Yang W, Li C, Zheng X, Lu Z, Guo C, et al. Fbxw7 is an independent prognostic marker and induces apoptosis and growth arrest by regulating YAP abundance in hepatocellular carcinoma. Mol Cancer 2014;13:110. 41. Kostenko S, Dumitriu G, Moens U. Tumour promoting and suppressing roles of the atypical MAP kinase signalling pathway ERK3/4-MK5. J Mol Signal 2012;7:9. 42. Perander M, Keyse SM, Seternes OM. New insights into the activation, interaction partners and possible functions of MK5/PRAK. Front Biosci (Landmark Ed) 2016;21:374-84. 43. Zheng M, Wang YH, Wu XN, Wu SQ, Lu BJ, Dong MQ, et al. Inactivation of Rheb by PRAK- mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1. Nat Cell Biol 2011;13:263-72. 44. Kim Y, Kim C, Son SM, Song H, Hong HS, Han SH, et al. The novel RAGE interactor PRAK is associated with autophagy signaling in Alzheimer's disease pathogenesis. Mol Neurodegener 2016;11:4. 45. Sun P, Yoshizuka N, New L, Moser BA, Li Y, Liao R, et al. PRAK is essential for ras-induced senescence and tumor suppression. Cell 2007;128:295-308. 46. Ronkina N, Johansen C, Bohlmann L, Lafera J, Menon MB, Tiedje C, et al. Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK. PLoS One 2015;10:e0136138. 47. Kapoor A, Yao W, Ying H, Hua S, Liewen A, Wang Q, et al. Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer. Cell 2014;158:185-97. 48. Kress TR, Cannell IG, Brenkman AB, Samans B, Gaestel M, Roepman P, et al. The MK5/PRAK kinase and Myc form a negative feedback loop that is disrupted during colorectal tumorigenesis. Mol Cell 2011;41:445-57. 49. Wang S, Xie F, Chu F, Zhang Z, Yang B, Dai T, et al. YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKvarepsilon-mediated phosphorylation. Nature immunology 2017;18:733-43. 50. Basu S, Totty NF, Irwin MS, Sudol M, Downward J. Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Molecular cell 2003;11:11-23. 51. Rosenbluh J, Nijhawan D, Cox AG, Li X, Neal JT, Schafer EJ, et al. beta-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis. Cell 2012;151:1457-73. 52. Levy D, Adamovich Y, Reuven N, Shaul Y. Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage. Molecular cell 2008;29:350- 61. 53. Sugihara T, Werneburg NW, Hernandez MC, Yang L, Kabashima A, Hirsova P, et al. YAP Tyrosine Phosphorylation and Nuclear Localization in Cholangiocarcinoma Cells Are Regulated by LCK and Independent of LATS Activity. Molecular cancer research 2018;16:1556-67.

Figure Legends

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 nM), or blebbistatin (Blebbi, 50 μM). D, Xenograft tumor growth assay of LATS1/2-wild type and LATS1/2-null RPE1 cells in immunocompromised mice. The left panel shows the gross and microscopic features (H&E, x40) 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 upper diagram shows the scheme of kinome siRNA library screening. The lower panel shows 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 Methods for details). Scale bars, (B, C, D, F) 15 μm; (E) 100 μm. Error bars indicate SEM (n = 5 LATS1/2-null RPE1 xenograft and n = 3 LATS1/2-wild type xenograft in D; *P < 0.05 and **P < 0.01, t test).

Figure 2. RNAi screening identified MK5 as a novel target for YAP/TAZ suppression. A, The left panel shows immunofluorescence micrographs of LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hr. 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 hr. C, qRT-PCR analysis of YAP/TAZ target gene expression in LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hr. D, Activity of a luciferase reporter with TEAD binding sites in LATS1/2- null RPE1 cells transfected with control or MK5 siRNAs for 48 hr. E, Relative cell viability of LATS1/2-null RPE1 cells transfected with MK5 siRNA compared to those with control siRNA. F, Phase contrast images of LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for

72 hr in 3D matrigel culture. G, The left panel shows Ki-67 immunofluorescence staining of LATS1/2-null RPE1 cells transfected with control or MK5 siRNAs for 48 hr. 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 to those with control siRNA for 48 hr in transwell migration assay. I, The left panel shows 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 hr. 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 hr. 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 hr. Scale bars, (A, G, I) 15 μm; (F) 45 μm. Error bars indicate SEM (n = 3 independent experiments; *P < 0.05 and **P < 0.01, t test).

Figure 3. Inhibition of MK5 suppresses the growth of YAP-driven tumors. A, Immunoblots of the indicated proteins in malignant mesothelioma (MM; MSTO-211H and H2373) and uveal melanoma (UVM; 92.1) cells transfected with control or MK5 siRNAs for 72 hr. B, Relative cell viability of YAP-driven MM and UVM cells transfected with MK5 siRNA compared to those with control siRNA for 72 hr and 96 hr. C, Phase contrast images of YAP-driven MM and UVM cells in 3D matrigel culture. Cells were transfected with control or MK5 siRNA for 72 hr and further incubated for 48 hr. 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 hr. F, Immunoblots of the indicated proteins in LATS1/2- null RPE1 cells treated with DMSO or H89 (10 μM) for 6 hr. G, Cell viability assay of PLX4032‐ resistant SKMEL28 cells treated with PLX4032 (PLX, 2 μM), H89 (10 μM), or their combinations for 72 hr. H, Immunoblots of the indicated proteins in PLX4032-resistant SKMEL28 cells treated with PLX4032 (PLX, 2 μM), H89 (10 μM), or their combinations for 24 hr. 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. The left panel shows the gross features of LATS1/2-null RPE1 xenograft. The graph shows tumor growth. J, The left panel shows the H&E staining of a xenograft tumor obtained from the experiment presented in (I). Mitotic cells are indicated by arrows. The graph is the quantification of mitotic

cells at 10 high power fields (HPF) by H&E 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, (C) 75 μm; (I) 1 mm; (J, K, L) 30 μm. Error bars indicate SEM (n = 3 independent experiments; n = 4 mice; *P < 0.05 and **P < 0.01, t test).

Figure 4. MK5 is a potential therapeutic target for YAP-driven cancers. A, The left panel shows 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-value < 0.05 and absolute log2 (fold change) ≥ 2. Right panel is the 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 were 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. The right panel shows 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), and lower grade glioma (TCGA-LGG). F, Representative figures of YAP and MK5 immunohistochemical staining in human uveal melanoma (UVM) tissues. Scale bar, 5 μm. The tables show the expression of MK5 and the activity of YAP in 80 primary and 16 metastatic UVM samples.

Figure 5. MK5 physically interacts with YAP and protects proteasomal degradation of YAP. A, Co-immunoprecipitation analysis of YAP and MK5 with lysates from RPE1 cells stably expressing FLAG-YAP vector. IP and IB denote immunoprecipitation and immunoblot, respectively. B, Co-immunoprecipitation 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 co-localization of exogenous YAP and MK5 in RPE1 cells. The lower panels are magnified view of the boxed area. Scale bar, 5 μm. F, Schematic representations of the C-terminal MK5 deletion constructs. G and H, Co-immunoprecipitation 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, K, and L, Co-immunoprecipitation analysis of MK5 and the indicated YAP deletion constructs with lysates from RPE1 cells stably expressing V5-MK5.

Figure 6. MK5-YAP interaction counteracts CK1δ/ε-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 hr, and treated with Cycloheximide (CHX, 100 μg/ml). B and C, 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 hr, and further treated with Bafilomycin A (Baf A, 10 nM) or MG132 (10 μM) for 6 hr. D, Ubiquitination analysis of precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with HA-Ubiquitin and V5-MK5 expression vectors. Cells were treated with MG132 (10 μM) for 6 hr after 24 hr of transfection. IP and IB denote immunoprecipitation and immunoblot, respectively. E, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells transfected with control or

CK1δ/ε siRNAs for 72 hr. F, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with PF670462 (100 nM, 500 μM, or 1 μM) for 24 hr. G, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells transfected with control, MK5, or MK5 and CK1δ/ε siRNAs for 48 hr. H, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells transfected with empty, CK1ε, or CK1ε and MK5 vectors for 24 hr. I, Ubiquitination analysis of precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with the indicated plasmids for 24 hr, and treated with MG132 (10 μM) for 6 hr. J, Immunoblots of the indicated proteins in LATS1/2- null RPE1 cells transfected with control, MK5, or MK5 and β-TRCP siRNAs for 48 hr. K, Immunoprecipitation assay of endogenous β-TRCP with precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with empty or V5-MK5 vector.

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 hr. B, Ubiquitination analysis of precipitated YAP. RPE1 cells stably expressing FLAG-YAP were transfected with the indicated plasmids for 24 hr, and treated with MG132 (10 μM) for 3 hr. C, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with DMSO or MK2/MK3/MK5 inhibitor (PF3644022, 50 μM) for 48 hr. D, Immunoblots of the indicated proteins in LATS1/2-null RPE1 cells treated with DMSO,

PF3644022 (50 μM, 48hr), or PF3644022 (50 μM, 48hr) and PF670462 (1 μM, 12hr). E, Co- immunoprecipitation 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 μM) for 24 hr. F, Immunoblots of the indicated proteins in RPE1 cells stably expressing FLAG-YAP-5SA transfected with control or MK5 siRNAs for 48 hr. G, Immunofluorescence images of RPE1 cells stably expressing FLAG-YAP-5SA transfected with control or MK5 siRNAs for 48 hr. H, Co-immunoprecipitation 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, (G) 12 μm.

MK5 regulates YAP stability and is a molecular target in YAP-driven cancers

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

Cancer Res Published OnlineFirst October 2, 2019.

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

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/10/02/0008-5472.CAN-19-1339.DC1

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

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