Targeting Hippo-Dependent and Hippo-Independent YAP1 Signaling for the 2 Treatment of Childhood Rhabdomyosarcoma 3

Author Manuscript Published OnlineFirst on April 30, 2020; DOI: 10.1158/0008-5472.CAN-19-3853 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Targeting Hippo-dependent and Hippo-independent YAP1 signaling for the 2 treatment of childhood rhabdomyosarcoma 3

4 Katherine K. Slemmons1, Choh Yeung2, Joshua T. Baumgart2, Jhazeel O. Martinez Juarez1, 5 Amy McCalla2, Lee J. Helman1,3

6 7

8 1Department of Hematology/Oncology, Children’s Hospital Los Angeles, Los Angeles, 9 California; 2Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, 10 Bethesda, MD; 3Departments of Pediatrics and Medicine, University of Southern California, Los 11 Angeles, California 12

14 15 16 17 18 ______19 Running title 20 Targeting YAP1 signaling in RMS 21 Key words 22 Rhabdomyosarcoma, YAP1, YES1, DNA methyltransferase inhibitors 23 Corresponding author 24 Lee J. Helman, (323) 361-6058 25 Reprint requests 26 Send reprint requests to Lee J. Helman, 4650 Sunset Blvd MS#57, Los Angeles, CA 90027; 27 [email protected] 28 Disclosure of Potential Conflicts of Interest 29 L.J. Helman is a consultant for Boehringer Ingelheim, Roche Bioscience, SpringWorks, and VujaDe, 30 holds stock in AstraZeneca and Viela Bio, and has cooperative research and development 31 agreements with Boehringer Ingelheim and AbbVie. No potential conflicts of interest were disclosed 32 by the other authors. 33

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 30, 2020; DOI: 10.1158/0008-5472.CAN-19-3853 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Abstract

2 Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, yet

3 patients with metastatic or recurrent disease continue to do poorly, indicating a need for new

4 treatments. The SRC family tyrosine kinase YES1 is upregulated in RMS and is necessary for

5 growth, but clinical trials using single agent Dasatinib, a SRC family kinase inhibitor, have failed

6 in sarcomas. YAP1 (YES-associated protein) is highly expressed in RMS, driving growth and

7 survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to

8 pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of

9 RMS with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo-activators RASSF1 and

10 RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing

11 inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased RMS cell growth

12 and increased apoptosis and differentiation, an effect partially rescued by expression of

13 constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are in part due to

14 Hippo-dependent inhibition of YAP1. Additionally YES1 and YAP1 interacted in the nucleus of

15 RMS cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention

16 of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a

17 Hippo-independent manner. Combined treatment with DNMTi and Dasatinib targeted both

18 Hippo-dependent and Hippo-independent regulation of YAP1, ablating RMS cell growth in vitro

19 and trending towards decreased tumor growth in vivo. These results show that the mechanisms

20 regulating YAP1 in RMS can be inhibited by combinatorial therapy of DNMTi and Dasatinib,

21 laying the groundwork for future clinical investigations.

23 Significance

24 This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and

25 identifies a combination therapy to target these pathways in the childhood tumor

26 rhabdomyosarcoma.

1 Introduction

2 Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. While

3 outcomes for patients with localized disease have improved, patients with metastatic or recurrent

4 disease continue to do poorly, therefore new treatments are needed (1,2). The two most common

5 subtypes of RMS, embryonal (eRMS) and alveolar (aRMS), are phenotypically and molecularly

6 distinct but often treated with the same modalities. Standard of care treatment includes a

7 combination of surgery, radiation, and a backbone of cytotoxic chemotherapies made up of

8 vincristine, actinomycin D, and cyclophosphamide but does not include any targeted therapies.

9 eRMS is characterized by genomic instability with high rates of copy number alternations, single

10 nucleotide variations and up to one third have alterations in the RAS signaling pathway (3,4). On

11 the other hand, aRMS is characterized by a distinct PAX3/7-FOXO1 (PF) chromosomal

12 translation creating a fusion transcription factor. PF expression results in widespread

13 transcriptional disruption and upregulation of oncogenic pathways including FGFR4 and IGF

14 signaling (5). The presence of the PF fusion gene portends a worse prognosis with a 5 year

15 survival rate of 52% for localized disease, and 19% when metastatic (6).

16 The YAP1 protein was initially discovered as the YES-associated-protein. YES1, a SRC

17 family tyrosine kinase (SFK), has been shown to be upregulated in RMS and drive tumor cell

18 proliferation (7). Although the YES1 kinase is targetable using Dasatinib and other kinase

19 inhibitors, to date it has not proven to be effective in clinical trials in RMS (8). However, the

20 relationship between YES1 and YAP1 in RMS has not been studied.

21 YAP1 was discovered to be the terminal oncoprotein in the Hippo tumor suppressor

22 pathway, a developmental pathway that regulates organ size and tissue regeneration. MST1/2,

23 the mammalian ortholog of Hippo, is a tumor suppressor that initiates a phosphorylation cascade

24 through LATS1/2 that results in the phosphorylation of YAP1 and its homolog TAZ at conserved

1 serine residues. Once phosphorylated, YAP1 is sequestered in the cytoplasm and can be

2 degraded. MST1/2 is regulated upstream by many inputs including cell-to-cell contact, actin

3 cytoskeleton, and the RASSF family of scaffold proteins. When MST1/2 is inactive, YAP1 is not

4 phosphorylated and translocates to the nucleus where it functions as a transcriptional co-

5 activator, binds the TEAD family of transcription factors, and initiates transcription of pro-growth

6 and anti-apoptotic genes (9). Hippo signaling is dysregulated in many cancer types through

7 epigenetic silencing of Hippo-activator RASSF1, MST1/2 or LATS1/2, or through general

8 upregulation of YAP1 and/or TAZ expression. YAP1 is highly expressed in human RMS tumors

9 where it has been shown to promote proliferation, survival, and stemness, and inhibit myogenic

10 differentiation (10–12). While YAP1 is an ideal therapeutic target in cancer, a specific YAP1

11 inhibitor has yet to reach the clinic. We aimed to identify a clinically-relevant therapy to inhibit

12 YAP1 in RMS.

13 Here we demonstrate that treatment with a DNA methyltransferase inhibitor (DNMTi) can

14 inhibit YAP1 in a Hippo-dependent manner through alteration of RASSF family expression

15 (upstream MST1/2 regulators). Furthermore, YAP1 is regulated in a Hippo-independent manner

16 through YES1 and this regulation can be inhibited through treatment with a SRC family kinase

17 inhibitor, Dasatinib. Lastly, a combined treatment results in ablation of cell growth and induction

18 of apoptosis. These data suggest a potential therapeutic strategy using the combination of

19 DNMTis and SFK inhibitors for the treatment of recurrent/refractory RMS, particularly the alveolar

20 RMS subtye.

21 Materials and Methods

22 Generation of Cell Lines and Constructs

23 Human RMS cell lines RD (eRMS) and Rh30 (aRMS) were obtained from ATCC. Rh28 and

24 RMS559 were obtained from Dr. Javed Khan (NCI, Bethesda, MD). Rh36 eRMS cell line was

1 obtained from Dr. Maria Tsokos (Beth Israel Medical Center, Boston, MA). Rh41 aRMS cell line

2 was obtained from Dr. Peter Houghton (St Jude's Children's Research Hospital, Memphis, TN).

3 Cell line authentication was performed in November 2018 using STR analysis (Promega

4 PowerPlex 16 HS System) conducted by the University of Arizona Genetics Core. The cell lines

5 were cultured in RPMI-1640, 100 units/ml penicillin and 100 μg/ml streptomycin, 2 mM glutamine,

6 and 10% heat inactivated fetal bovine serum at 37ºC in an atmosphere of 5% CO2. Lentivirus

7 shYES clones 9 and 11 were obtained from Sigma (TRCN0000001609 and TRCN0000001611,

8 respectively) and used as previously described (7). YAPS127A cell line was constructed by

9 transfecting Rh30 cells with pCDNA4/HismaxB-YAP1-S127A (Addgene plasmid # 18988) and

10 vector control cell was transfected with pCDNA4/TO (Thermo Fisher) using Nucleofector (Lonza)

11 program B032, buffer V, using 2x106 cells, and 2µg of plasmid. Cells were batch selected using

12 zeocin (Thermo Fisher). YEST348I cell line was constructed by stably expressing YES1 (T348I)

13 in pLX303 (Addgene plasmid # 42564) or vector control pLX304 (Addgene plasmid # 25890) in

14 Rh30 cells using established lentiviral methods (11). Cells were batch selected using blasticidin

15 (Invitrogen).

16 IncuCyte Growth Curves and Apoptosis Assays

17 For growth curves, 1000-2000 RMS cells per well were plated in 96-well plates (CytoOne) with 5-

18 10 wells per condition. 24 hours after plating cells were treated with 0.5µM SGI-110, 0.5µM

19 Dasatinib, or combination of SGI-110 and Dasatinib at 0.25µM or 0.5µM and loaded into the

20 IncuCyte S3 (Essen BioScience). Images were taken every 4 hours for 7 days and percent

21 confluence was calculated. For cleaved caspase 3/7 activity, cells were treated with SGI-110 for

22 5 days, then media was aspirated and fresh media containing SGI-110 and the IncuCyte

23 Caspase-3/7 Green Apoptosis Assay Reagent (at 1:1000 dilution) were added. Images were

24 obtained every 2 hours for 3 days.

2 Quantitative Real Time PCR and Methylation-specific PCR

3 RNA and DNA extractions were performed on the KingFisher (ThermoFisher) per manufacturer’s

4 recommendations. Reverse transcription was performed with the iScript Select cDNA synthesis

5 kit (Bio-Rad). Quantitative Real Time PCR (qRT-PCR) was performed with the PowerUp SYBR

6 Green Master Mix (Thermo Fisher) per the manufacturer’s protocol. For Methylation-specific PCR

7 (MS-PCR), bisulfite conversion was performed with EZ DNA Methylation-lightning kit (Zymo) and

8 RASSF1 MS-PCR was done with the CpG WIZ RASSF1A Amplification kit (Millipore). Primer

9 sets are listed in Supplemental Table 1.

10 Immunoblots

11 0.5x106 to 1x106 cells were plated in 10-cm plates and treated with SGI-110, Dasatinib, or

12 combination for 48hrs to 5 days. Cell were harvested with RIPA cell lysis buffer (Cell Signaling)

13 plus protease and phosphatase inhibitors (Thermo Fisher). The following antibodies were used

14 for immunoblotting: anti-Cleaved PARP (Cell Signaling #5625, 1:1000), anti-RASSF1 (Abcam

15 #ab23950, 1:1000), anti-RASSF5 (Sigma #N5912, 1:200), anti-RASSF4 (Novus Bio #NBP1-

16 89249, 0.4µg/ml), anti-P-YAP1 Ser127 (Cell Signaling #13008, 1:1000), anti-P-Src Family Tyr416

17 (Cell Signaling #6943, 1:1000), anti-YES1 (Cell Signaling #3201, 1:500), anti-YAP (Cell Signaling

18 #14074, 1:1000), anti-Tubulin (Proteintech #66031-1-Ig, 1:2000), anti-GAPDH (Cell Signaling

19 #5174, 1:1000), anti-Vimentin (Cell Signaling #5741, 1:1000), anti-Histone H3 (Cell Signaling

20 #4499, 1:2000), anti-MEK1/2 (Cell Signaling #8727, 1:1000), anti-AKT (Cell Signaling #4685,

21 1:1000), anti-P-AKT Ser473 (Cell Signaling #4060, 1:2000), anti-ERK1/2 (Cell Signaling #4695,

22 1:1000). Densitometry was performed using ImageJ (NIH) on 3 biological replicates and all values

23 were normalized to the loading control.

1 Proximity Ligation Assays (PLA)

2 PLA was performed to detect co-localization of YES1 and YAP1 using the Duo-Link In Situ Orange

3 kit (Sigma). RMS cells were plated at 500 cells per well on a 384 well glass bottom plate in 50ul

4 volume and incubated for 48 hours. The cells were fixed by adding 50ul of 4% paraformaldehyde

5 (Santa Cruz Biotechnology) per well and allowed to fix for 20 minutes at 4ºC. The fixed cells were

6 washed twice with PBS and blocked/permeabilized with 50ul/well of blocking solution (5% donkey

7 serum, 0.3% Triton X100 in PBS) for one hour at 37ºC. Blocked/permeabilized cells were

8 incubated in primary antibodies overnight with anti-YAP1 (Abcam #ab39361) and/or anti-YES1

9 (Wako #013-14261) at 1:50 dilution. Labeling, ligation, and amplification reactions were

10 performed according to the Duo-Link kit. Visualization and analysis of the labeled cells were done

11 using an Opera Phenix system (Perkin-Elmer).

12 Luciferase Assays

13 LATS biosensor plasmids pCDNA3.1neo-14-3-3-CLuc (Addgene plasmid #107611) and

14 pCDNA3.1neo-NLucYAP15 (Addgene plasmid #107610) were gifts from Xiaolong Yang (Queen's

15 University, Kingston, Ontario, Canada). pcDNA3 Lats1 (Nigg HS189) was a gift from Erich Nigg

16 (Addgene plasmid # 41156). The assay was performed as previously described with some

17 modifications (13,14). Fugene 6 (Promega) was used to transfect 100ng of each plasmid for the

18 Rh30 cells and 200ng for the RD cells in a 96 well plate. For an assay positive control 400ng

19 pcDNA3 LATS1 was transfected at the same time. Living cell luciferase was measured on the

20 Varioskan LUX multimode microplate reader (ThermoFisher) after a 10 min incubation with

21 0.05mg D-Luciferin (Promega). Luciferase assays were replicated in three individual experiments

22 with each containing five replicates per condition.

1 Drug Studies

2 DNMTi Guadecitabine or SGI-110 was obtained from AdooQ and MedChemExpress and

3 resuspended in DMSO at 10mM for in vitro studies or in PBS at 5mM for in vivo studies. Dasatinib

4 was obtained from SelleckChem and resuspended in DMSO at 10mM for in vitro studies or in 4%

5 DMSO + 30% PEG 300 + 5% Tween 80 in ddH2O for in vivo studies.

6 RNASeq and EPIC array

7 For RNAseq, RNA samples were collected from RD or Rh30 cells treated with 0.5µM SGI-110,

8 Dasatinib, or combination for 5 days in biological triplicates. The library prep and sequencing was

9 performed by the Single Cell, Sequencing, and CyTOF (SC2) Core Laboratory at the Saban

10 Research Institute of Children's Hospital Los Angeles. Transcriptome libraries were prepared from

11 total RNA using the Illumina TruSeq Stranded mRNA Library Prep kit following manufacturer’s

12 protocol, which allows for standardized mRNA isolation from total RNA, first- and second-strand

13 cDNA synthesis, end-repair, A-base addition, and adapter ligation. Libraries were PCR amplified

14 and paired-end cluster generation was performed using the Illumina’s Nextseq 500 on a High-

15 Output Sequencing Reagent Kit v2 (2 x 75bp). Quality control and adapter trimming were

16 performed using trim_galore (v0.4.2) with default parameters

17 (https://github.com/FelixKrueger/TrimGalore). Reads were aligned to the GRCh38 reference

18 genome and transcriptome using HISAT2 (v2.1.0) (15), and transcript quantification was

19 performed using feature Counts (v1.5.1) (16). Differential expression analysis was performed

20 using the ‘DESeq2’ R package (v1.16.1) (17), and a rank score calculated as -log10(q-

21 val)*sign(log2FoldChange) was used as input to the GSEA Preranked tool for pathway analysis

22 (18). Data were analyzed through the use of Ingenuity Pathway Analysis (IPA) (QIAGEN

23 Inc.,https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) (19).

1 For the Illumina Infinium HumanMethylationEPIC (EPIC) DNA methylation assay, genomic DNA

2 (1000 ng) was bisulfite converted using the Zymo EZ DNA methylation kit (Zymo Research, Irvine,

3 CA) according to the manufacturer’s recommendations. The amount of bisulfite-converted DNA

4 as well as the completeness of bisulfite conversion for each sample were assessed using a panel

5 of MethyLight-based real-time PCR quality control assays as described previously (20). Bisulfite-

6 converted DNAs were then repaired using the Illumina Restoration Kit as recommended by the

7 manufacturer. The repaired DNA was used as a substrate for the Illumina EPIC BeadArrays, as

8 recommended by the manufacturer and first described in Moran, 2016 (21). After the chemistry

9 steps, BeadArrays were scanned and the raw signal intensities are extracted from the *.IDAT files

10 using the ‘noob’ function in the minfi R package. The ‘noob’ function corrects for background

11 fluorescence intensities and red-green dye-bias as described by Triche et al (22). The beta value

12 was calculated as (M/(M+U)), in which M and U refer to the (pre-processed) mean methylated

13 and unmethylated probe signal intensities, respectively. Measurements in which the fluorescent

14 intensity is not statistically significantly above background signal (detection p value > 0.05) were

15 removed from the data set. All the RNAseq and EPIC array data were submitted to GEO (Gene

16 Expression Omnibus) and assigned repository #GSE147240 and #GSE147246, respectively.

17 Immunofluorescence

18 YAP1 immunofluorescence (IF; Cell Signaling Technology #14074, 1:100) was performed

19 according to the manufacturer’s protocol except for the addition of a permeabilization step with

20 0.3% Triton X in PBS pH 7.4 before blocking step.

21 Xenograft Studies

22 2x106 luciferase labeled (pGL4.18 CMV-Luc, Addgene plasmid # 100984) Rh30 cells were

23 resuspended in sterile HBSS and injected intramuscularly into the right gastrocnemius of female

24 4-6 week old SCID/beige mice. Treatment began at Day 10 after injection when tumors were

25 detectable on bioluminescence imaging and mice were randomized into treatment groups.

1 Dasatinib was given at 100mg/kg three times a week by oral gavage and DNMTi Guadecitabine

2 was given at 2mg/kg five days in a row on 14 day cycles by subcutaneous injections. Mice were

3 observed twice weekly for evidence of malaise, weight loss or inability to ambulate normally, and

4 the limbs were measured with calipers. Tumor volume was calculated by the following formula: V

5 (mm3)= (D x d2)/6 x 3.14, where D is the longest tumor axis and d is the shortest tumor axis as in

6 previous studies (23). Mice were sacrificed at Day 33 post tumor cell injection due to weight loss

7 in the combination treated group. Tumor weight was measured by subtracting the weight of the

8 non-tumor bearing leg from the weight of the tumor bearing leg. Tumors were preserved in

9 RNAlater (Qiagen) for PCR analysis or formalin-fixed for IHC. Studies were approved by CHLA’s

10 Institutional Animal Care and Use Committee (IACUC).

11 Statistical Analysis

12 Statistical analysis was performed using GraphPad Prism (GraphPad). Unless otherwise noted,

13 data is presented as the mean and SE. One-way ANOVA, two-way ANOVA, and unpaired T-test

14 were used as appropriate. P values were considered significant at *, P< 0.05; **, P<0.01; ***,

15 P<0.001; and ****, P<0.0001.

16 Results

17 We analyzed the effect of treatment with a DNA methyltransferase inhibitor, SGI-110 also

18 known as Guadecitabine, on the growth of RMS cell lines using live cell imaging. DNMTi treatment

19 markedly decreased RMS cell growth in both aRMS and eRMS cells (Fig. 1A and Supplemental

20 Fig. 1A). To determine if DNMTi treatment causes RMS cell death we analyzed apoptosis by a

21 cleaved caspase 3/7 fluorescence reporter and by immunoblots of cleaved PARP. DNMTi

22 treatment markedly increased apoptosis in aRMS cells and modestly increased apoptosis in

23 eRMS cells (Fig. 1B,C and Supplemental Fig. 1B,C). The aRMS cells were more sensitive to

1 DNMTi treatment exhibiting a 5X increase in the percentage of cleaved caspase 3/7 positive cells

2 compared to control while the eRMS cells only had a 2X increase in apoptosis.

3 After treatment with a DNMTi we also observed a striking phenotypic change in a

4 percentage the RMS cells. In addition to the obvious apoptotic cells, we observed large flat

5 elongated cells that resembled skeletal muscle myoblasts (Fig. 1D). qRT-PCR for MYF6, a

6 myogenic factor involved in the later stages of skeletal muscle differentiation, showed a large

7 increase in MYF6 expression (Fig.1E). These data suggest DNMTi treatment promotes changes

8 towards myogenic differentiation which may contribute to the growth inhibitory effects of drug

9 treatment. The decrease in growth may also be due to cellular senescence which was not

10 explored in this study but has been observed after Hippo pathway activation in RMS (11,24).

11 To validate that DNMTi treatment could successfully demethylate the RMS genome, we

12 analyzed changes in whole genome DNA methylation by the Illumina EPIC methylation array

13 which analyzes the methylation status of over 850,000 sites in the genome. The EPIC array

14 revealed that DNMTi treatment for 5 days was sufficient to cause widespread DNA demethylation

15 in both the Rh30 and RD cells (Fig. 2A). To determine the effect of genome demethylation on

16 gene expression we performed RNAseq on Rh30 and RD cells after DNMTi treatment. RNAseq

17 revealed changes in expression of the RASSF family, regulators of Hippo (MST1/2). RASSF1, 5,

18 6, which have roles in activating MST1/2, were elevated at the mRNA and protein level (Fig. 2C,

19 D and Supplemental Fig. 2A). On the other hand, RASSF4, which has been shown to inhibit

20 MST1/2 in aRMS (11), decreased in expression (Fig. 2C,D and Supplemental Fig. 2A). YAP1

21 itself also decreases at the RNA level and several myogenic differentiation genes including MYF5,

22 MYF6 increase in accordance with our observations in Fig. 1D, E (Fig. 2B). FGFR4, another

23 oncogenic signaling pathway in RMS (25,26), was also significantly decreased (Fig. 2B). These

24 data demonstrate that genes relevant to RMS tumorigenesis are altered in expression after

25 DNMTi treatment. Furthermore, we confirmed by MS-PCR that the promoters of RASSF1 and

1 RASSF5 are ordinarily methylated in aRMS cells and eRMS cells but become partially

2 demethylated after DNMTi treatment (Fig. 2E and Supplemental Fig. 2B). Interestingly, RASSF4

3 was also partially demethylated as analyzed on the EPIC methylation array (Fig.2F). These data

4 demonstrate DNMTi treatment in RMS cells results in upregulation of tumor suppressor RASSF

5 family members 1,5,6 and downregulation of pro-growth RASSF4.

6 To determine the effect of changes in RASSF expression on downstream Hippo pathway

7 signaling we performed a biosensor for LATS1/2 activity. Luciferase is expressed when YAP1 is

8 phosphorylated at S127 by LATS1/2 and is sequestered by 14-3-3 (13). There is a baseline

9 amount of luminescence or LATS1/2 activity in RMS which then doubles after DNMTi treatment

10 suggesting DNMTi treatment activates canonical Hippo signaling (Fig. 3A). This increase in

11 LATS1/2 activity after DNMTi treatment is comparable to the increase seen after transient

12 expression of LATS1 in these cells (Fig.3A). Furthermore, by immunoblot DNMTi treatment

13 results in an increase in inactive P-YAP1 (Fig. 3B) and the DNMTi-induced growth inhibition can

14 be partially rescued by overexpression of a constitutively active YAPS127A (a mutation that

15 prevents YAP1 serine phosphorylation and cytoplasmic localization), suggesting that the growth

16 inhibition after DNMTi treatment is in part due to YAP1 inactivation (Fig. 3C, D). DNMTi treatment

17 also decreased both YES1 expression and Y416 phosphorylation, a marker of YES1 activity (Fig.

18 3B). These data suggest DNMTi treatment can inhibit both YAP1 and YES1 signaling. Overall,

19 these data demonstrate that DNMTi treatment can inhibit YAP1 signaling in a Hippo-dependent

20 manner through modulation of RASSF family expression and can decrease YES1 activation.

21 We decided to focus more closely on the aRMS subtype in the rest of the study since the

22 aRMS cells were more sensitive to DNMTi treatment. To further investigate the connection

23 between YES1 and YAP1, we performed proximity ligation assays (PLA) and discovered that

24 YES1 and YAP1 interact in the nucleus of Rh30 aRMS cell line as in other cell types (Fig. 4A).

25 Furthermore, shRNA knockdown of YES1 promotes YAP1 cytoplasmic localization and

1 decreases expression of the YAP1 target genes, suggesting a role for YES1 in regulating YAP1

2 cellular localization in RMS (Fig. 4B, C). Similarly, treatment with the SRC family kinase inhibitor

3 Dasatinib decreases expression of the YAP1 target genes (Fig. 4D) and results in YAP1

4 cytoplasmic localization shown by YAP1 immunofluorescence (Fig. 4E). The localization of YAP1

5 to the cytoplasm after Dasatinib treatment can be rescued by expression of a Dasatinib resistant

6 YES1 T348I (Fig. 4E, Supplemental Fig. 3) suggesting YES1 activity is necessary to control

7 YAP1 localization. Expression of YEST348I was confirmed by immunoblot (Fig.4F). Together

8 these data propose a dual regulation of YAP1 in RMS through Hippo-dependent and Hippo-

9 independent (via YES1) mechanisms.

10 To target both mechanisms of YAP1 regulation we evaluated combination treatment of

11 DNMTi and Dasatinib in RMS cells in vitro. The combination ablated cell growth, showing greater

12 growth inhibition than either drug alone (Fig. 5A and Supplemental Fig. 4A). Combination

13 treatment also resulted in a marked increase in apoptosis, shown by immunoblots for cleaved

14 PARP (Fig. 5B). These data demonstrate DNMTi and Dasatinib combination treatment has

15 greater activity than each individual drug which results in an ablation of RMS cell growth.

16 To analyze possible mechanisms of this combination, we used Ingenuity Pathway

17 Analysis (IPA) to compare the RNAseq transcriptional profiles of Rh30 and RD cells treated with

18 the combination compared to DNMTi alone treated cells. IPA analysis uses a knowledge network

19 to determine the canonical pathways predicted to be activated or inactivated in the dataset. The

20 top canonical pathways enriched in the combination group showed a variety of pathways including

21 several pathways regulating the actin cytoskeleton (Supplemental Table 2). Of interest were the

22 inactivation of NF-kB signaling and the activation of PTEN signaling because of their roles in cell

23 proliferation and survival. When we analyzed signaling downstream of PTEN, we saw a near

24 elimination of AKT signaling (analyzed by P-AKT) in the aRMS cell lines while this decrease in P-

25 AKT was not seen in the eRMS cell lines (Fig. 5C, top). This is in line with the higher rates of

1 apoptosis seen in the aRMS cell lines. While multiple pathways are likely altered with DNMTi and

2 Dasatinib treatment, we show at least two possible pathways (NF-kB and PTEN) that are altered

3 by the combination treatment and may contribute to the activity observed.

4 Because of the striking inhibition of cell growth after combination treatment we pursued

5 the combination treatment in orthotopic xenografts in immunocompromised mice. Here we

6 focused on the aRMS subtype since the aRMS cells were more sensitive to DNMTi treatment

7 than the eRMS cells (Figure 1). We generated orthotopic aRMS xenografts from the Rh30 cell

8 line and began treatment once tumors were detectable on imaging. DNMTi treatment alone

9 significantly decreased tumor growth over time as measured by calipers (Fig. 6A). Meanwhile,

10 Dasatinib treatment alone did not significantly inhibit tumor growth due to variability, as has been

11 seen in previous studies (7). Unfortunately, toxicity arose in the combination group after the

12 second round of DNMTi treatment, so the study was terminated early. However, there was a trend

13 towards decreased tumor growth in the combination treated group, but it did not reach significance

14 due to variability (Fig. 6A). Additionally, tumor weight was significantly smaller in the combination

15 treated group (Fig. 6B). Like in vitro, DNMTi treatment also results in an upregulation in

16 expression of the myogenic differentiation gene MYF6, albeit a smaller magnitude of change is

17 observed in vivo (Fig. 6C). However, H&E analysis did not show any obvious differences between

18 the groups (Supplemental Figure 5). Together these data are suggestive that combination

19 treatment could be effective in vivo but further exploration is needed.

20 In conclusion, in RMS cells YAP1 is regulated by both Hippo-dependent and Hippo-

21 independent signaling. DNMTi treatment leads to widespread genomic demethylation including

22 at the RASSF1 and RASSF5 tumor suppressor sites. These alterations in RASSF expression

23 lead to activation of canonical Hippo signaling and inactivation of YAP1. In a Hippo-independent

24 manner, YES1 associates with YAP1 and promotes YAP1 nuclear localization. This regulation of

25 YAP1 can be inhibited with Dasatinib treatment. Lastly targeting both mechanisms of YAP1

1 regulation by combination DNMTi and Dasatinib treatment ablates RMS cell growth laying the

2 foundation for clinical investigations of this combination treatment (Fig. 6D).

3 Discussion

4 Here we describe one mechanism by which DNMTi treatment inhibits RMS cell growth-

5 through Hippo-dependent YAP1 inhibition. However, DNMTis are not targeted or specific to

6 YAP1. As shown, DNMTi treatment causes widespread genomic demethylation and

7 transcriptional changes and future studies would be needed to fully examine the multitude of

8 possible mechanisms causing RMS growth inhibition. However, we hope this study further

9 emphasizes the need for a specific YAP1 inhibitor for cancer treatment, and further demonstrates

10 the utility of such a compound for RMS treatment. To date, a specific YAP1 inhibitor has not

11 reached the clinic despite continued research efforts (27). There has been continued effort in

12 identifying alternative approaches to inhibit YAP1 signaling such as the use of statins or GPCR

13 modulators to regulate the signaling upstream of YAP1 in particular cell types ((28) and reviewed

14 in (29)). Additionally, verteporfin (VP) was identified in a screen as an inhibitor of the interaction

15 between YAP1 and the TEAD family of transcription factors. However, subsequent studies

16 showed VP had off-target effects and significant solubility issues that limit its use clinically

17 (10,30,31). Here we suggest use of DNMTis as an alternative approach to YAP1 inhibition until a

18 specific modulator is available.

19 Interestingly, the RASSF1 promoter has been reported to be methylated in pediatric RMS

20 but not in adult RMS (32,33). This suggests a potential vulnerability of pediatric RMS to epigenetic

21 modifiers that can activate RASSF1 and other epigenetically silenced tumor suppressors and may

22 be one advantage of using a DNMTi. Cytosine analog DNMTis such as Guadecitabine are thought

23 to inhibit all DNMT isoforms through their incorporation into the genome. This is an advantage in

24 RMS as several isoforms including DNMT1 and DNMT3B have been shown to promote RMS

1 tumorigenesis (34,35). We see a significant effect of Guadecitabine treatment at inhibiting RMS

2 tumor growth in vivo as a single agent but the use of Guadecitabine in combination with other

3 treatments such as traditional chemotherapies, immunotherapy or other targeted therapies has

4 not been evaluated in RMS and warrants further exploration.

5 Lastly, this work lays the foundation for future clinical investigations of combination

6 Guadecitabine and Dasatinib treatment for RMS patients. Importantly both of these drugs have

7 been used in children and have minimal toxicities. Guadecitabine is currently being tested in over

8 23 active clinical trials and currently being evaluated in children with another type of sarcoma,

9 GIST (NCT03165721). Dasatinib is already FDA approved for use in children with chronic

10 myelogenous leukemia (CML) or Philadelphia chromosome positive ALL (36), and has been used

11 in RMS patients (NCT00464620, NCT03041701). Dasatinib is the preferred SFK inhibitor for RMS

12 because it has higher specificity for YES1, the highest expressed SFK in RMS (23). Furthermore,

13 a combination of imatinib (another SFK inhibitor) and decitabine (the earlier generation DNMTi)

14 was evaluated in a Phase II clinical trial in adults with CML and found to be safe and well tolerated

15 (37). Another case report showed combination azacitidine (another DNMTi) and Dasatinib to be

16 safe for the treatment of dual CML and myelodysplasia syndrome (38). These reports suggest

17 that, while we observed toxicity after combination treatment in our murine studies, combination

18 DNMTi and Dasatinib may be plausible in the clinic. Our results suggest aRMS patients would

19 benefit more from Guadecitabine treatment than eRMS patients. However, due to rarity of this

20 cancer likely clinical trials would include both subtypes of RMS.

21 Acknowledgements

22 The authors thank the Single Cell, Sequencing, and CyTOF (SC2) Core Laboratory at the Saban

23 Research Institute of Children's Hospital Los Angeles for their expertise and help in performing

24 and analyzing the RNAseq experiments presented and the USC Molecular Genomics Core for

1 their expertise and help in performing the Illumina EPIC methylation array. This research was

2 supported by a Rally Foundation for Childhood Cancer Research Fellowship (19FN14), a The

3 Saban Research Institute Research Career Development Fellowship (8030-RRI011550), and a

4 CHLA Core Pilot Project Program grant (RRI012015) (to K.K. Slemmons), and a USC/Norris

5 Comprehensive Cancer Center CORE Support grant (P30CA014089), the Nautica Endowment

6 Fund, and the Sarcoma Endowment Fund (to L.J. Helman).

7 References

8 1. Perkins SM, Shinohara ET, DeWees T, Frangoul H. Outcome for children with metastatic 9 solid tumors over the last four decades. PLoS ONE. 2014;9:e100396.

10 2. Pappo AS, Anderson JR, Crist WM, Wharam MD, Breitfeld PP, Hawkins D, et al. Survival 11 after relapse in children and adolescents with rhabdomyosarcoma: A report from the 12 Intergroup Rhabdomyosarcoma Study Group. J Clin Oncol. 1999;17:3487–93.

13 3. Shern JF, Chen L, Chmielecki J, Wei JS, Patidar R, Rosenberg M, et al. Comprehensive 14 genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a 15 common genetic axis in fusion-positive and fusion-negative tumors. Cancer Discov. 16 2014;4:216–31.

17 4. Chen X, Stewart E, Shelat AA, Qu C, Bahrami A, Hatley M, et al. Targeting oxidative stress 18 in embryonal rhabdomyosarcoma. Cancer Cell. 2013;24:710–24.

19 5. Cao L, Yu Y, Bilke S, Walker RL, Mayeenuddin LH, Azorsa DO, et al. Genome-wide 20 identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target 21 genes important for development and cancer. Cancer Res. 2010;70:6497–508.

22 6. Hibbitts E, Chi Y-Y, Hawkins DS, Barr FG, Bradley JA, Dasgupta R, et al. Refinement of risk 23 stratification for childhood rhabdomyosarcoma using FOXO1 fusion status in addition to 24 established clinical outcome predictors: A report from the Children’s Oncology Group. 25 Cancer Med. 2019;8:6437–48.

26 7. Yeung CL, Ngo VN, Grohar PJ, Arnaldez FI, Asante A, Wan X, et al. Loss-of-function 27 screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth. 28 Oncogene. 2013;32:5429–38.

29 8. Schuetze SM, Wathen JK, Lucas DR, Choy E, Samuels BL, Staddon AP, et al. SARC009: 30 Phase 2 study of dasatinib in patients with previously treated, high-grade, advanced 31 sarcoma. Cancer. 2016;122:868–74.

32 9. Yu F-X, Guan K-L. The Hippo pathway: regulators and regulations. Genes Dev. 33 2013;27:355–71.

1 10. Slemmons KK, Crose LES, Rudzinski E, Bentley RC, Linardic CM. Role of the YAP 2 Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma. PLoS ONE. 2015;10:e0140781.

3 11. Crose LES, Galindo KA, Kephart JG, Chen C, Fitamant J, Bardeesy N, et al. Alveolar 4 rhabdomyosarcoma-associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway 5 suppression. J Clin Invest. 2014;124:285–96.

6 12. Tremblay AM, Missiaglia E, Galli GG, Hettmer S, Urcia R, Carrara M, et al. The Hippo 7 transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal 8 rhabdomyosarcoma formation. Cancer Cell. 2014;26:273–87.

9 13. Azad T, Janse van Rensburg HJ, Lightbody ED, Neveu B, Champagne A, Ghaffari A, et al. 10 A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in 11 angiogenesis. Nat Commun. 2018;9:1061.

12 14. Azad T, Nouri K, Janse van Rensburg HJ, Hao Y, Yang X. Monitoring Hippo Signaling 13 Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor. 14 JoVE. 2018;58416.

15 15. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory 16 requirements. Nat Methods. 2015;12:357–60.

17 16. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for 18 assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30.

19 17. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA- 20 seq data with DESeq2. Genome Biol. 2014;15:550.

21 18. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set 22 enrichment analysis: A knowledge-based approach for interpreting genome-wide expression 23 profiles. Proceedings of the National Academy of Sciences. 2005;102:15545–50.

24 19. Krämer A, Green J, Pollard J, Tugendreich S. Causal analysis approaches in Ingenuity 25 Pathway Analysis. Bioinformatics. 2014;30:523–30.

26 20. Campan M, Weisenberger DJ, Trinh B, Laird PW. MethyLight. Methods Mol Biol. 27 2009;507:325–37.

28 21. Moran S, Arribas C, Esteller M. Validation of a DNA methylation microarray for 850,000 29 CpG sites of the human genome enriched in enhancer sequences. Epigenomics. 30 2016;8:389–99.

31 22. Triche TJ, Weisenberger DJ, Van Den Berg D, Laird PW, Siegmund KD. Low-level 32 processing of Illumina Infinium DNA Methylation BeadArrays. Nucleic Acids Res. 33 2013;41:e90.

34 23. Wan X, Yeung C, Heske C, Mendoza A, Helman LJ. IGF-1R Inhibition Activates a YES/SFK 35 Bypass Resistance Pathway: Rational Basis for Co-Targeting IGF-1R and Yes/SFK Kinase 36 in Rhabdomyosarcoma. Neoplasia. 2015;17:358–66.

1 24. Oristian KM, Crose LES, Kuprasertkul N, Bentley RC, Lin Y-T, Williams N, et al. Loss of 2 MST/Hippo Signaling in a Genetically Engineered Mouse Model of Fusion-Positive 3 Rhabdomyosarcoma Accelerates Tumorigenesis. Cancer Res. 2018;78:5513–20.

4 25. Crose LES, Etheridge KT, Chen C, Belyea B, Talbot LJ, Bentley RC, et al. FGFR4 blockade 5 exerts distinct antitumorigenic effects in human embryonal versus alveolar 6 rhabdomyosarcoma. Clin Cancer Res. 2012;18:3780–90.

7 26. Li SQ, Cheuk AT, Shern JF, Song YK, Hurd L, Liao H, et al. Targeting wild-type and 8 mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534). 9 PLoS ONE. 2013;8:e76551.

10 27. Crawford JJ, Bronner SM, Zbieg JR. Hippo pathway inhibition by blocking the YAP/TAZ– 11 TEAD interface: a patent review. Expert Opinion on Therapeutic Patents. 2018;28:867–73.

12 28. Sorrentino G, Ruggeri N, Specchia V, Cordenonsi M, Mano M, Dupont S, et al. Metabolic 13 control of YAP and TAZ by the mevalonate pathway. Nat Cell Biol. 2014;16:357–66.

14 29. Liu H, Du S, Lei T, Wang H, He X, Tong R, et al. Multifaceted regulation and functions of 15 YAP/TAZ in tumors (Review). Oncol Rep. 2018;40:16–28.

16 30. Lui JW, Xiao S, Ogomori K, Hammarstedt J, Little EC, Lang D. The Efficiency of Verteporfin 17 as a Therapeutic Option in Pre-Clinical Models of Melanoma. J Cancer. 2019;10:1–10.

18 31. Yu F-X, Zhang K, Guan K-L. YAP as oncotarget in uveal melanoma. Oncoscience. 19 2014;1:480.

20 32. Harada K, Toyooka S, Maitra A, Maruyama R, Toyooka KO, Timmons CF, et al. Aberrant 21 promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines. 22 Oncogene. 2002;21:4345–9.

23 33. Seidel C, Schagdarsurengin U, Blümke K, Würl P, Pfeifer GP, Hauptmann S, et al. Frequent 24 hypermethylation of MST1 and MST2 in soft tissue sarcoma. Mol Carcinog. 2007;46:865– 25 71.

26 34. Megiorni F, Camero S, Ceccarelli S, McDowell HP, Mannarino O, Marampon F, et al. 27 DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell 28 phenotype through inhibition of proliferation and induction of myogenic differentiation. 29 Oncotarget [Internet]. 2016 [cited 2020 Feb 23];7.

30 35. Ecke I, Petry F, Rosenberger A, Tauber S, Monkemeyer S, Hess I, et al. Antitumor Effects 31 of a Combined 5-Aza-2’Deoxycytidine and Valproic Acid Treatment on Rhabdomyosarcoma 32 and Medulloblastoma in Ptch Mutant Mice. Cancer Research. 2009;69:887–95.

33 36. Dasatinib Approved for Pediatric CML. Cancer Discov. 2018;8:OF2.

34 37. Oki Y, Kantarjian HM, Gharibyan V, Jones D, O’brien S, Verstovsek S, et al. Phase II study 35 of low-dose decitabine in combination with imatinib mesylate in patients with accelerated or 36 myeloid blastic phase of chronic myelogenous leukemia. Cancer. 2007;109:899–906.

1 38. Lang F, Wunderle L, Pfeifer H, Schnittger S, Bug G, Ottmann OG. Dasatinib and Azacitidine 2 Followed by Haploidentical Stem Cell Transplant for Chronic Myeloid Leukemia with 3 Evolving Myelodysplasia: A Case Report and Review of Treatment Options. Am J Case 4 Rep. 2017;18:1099–109.

7 Figure Legends

8 Figure 1. DNMTi treatment inhibits RMS cell growth and promotes aRMS cell death.

9 (A) IncuCyte live cell imaging over 7 days shows a decrease in Rh30 aRMS (top) and RD eRMS

10 (bottom) cell growth after 0.5µM DNMTi treatment. (B) Cleaved Caspase 3/7 activity is increased

11 after DNMTi treatment as measured by a fluorescent reporter on the IncuCyte in Rh30 aRMS

12 cells (top) and a small increase in RD eRMS cells (bottom). (C) Cleaved PARP is increased in

13 aRMS cells (left) by immunoblot after 5 days of treatment. (D) Representative images of cell

14 phenotype after 5 days of DNMTi treatment. Average percentage of elongated cells after DNMTi

15 treatment shown. (E) qRT-PCR for MYF6 demonstrates an increase in MYF6 expression after

16 DNMTi treatment in 5 RMS cell lines after 5 days of treatment. *, P<0.05; **, P<0.01; ***, P<0.001;

17 and ****, P<0.0001.

18 Figure 2. DNMTi treatment demethylates the genome and modulates RASSF family

19 expression.

20 (A) Frequency of Beta Values from the EPIC methylation array is plotted. 0=Unmethylated,

21 1=Fully Methylated. After DNMTi treatment (bottom) there is a shift of the peaks to the left

22 demonstrating widespread demethylation. (B) RNAseq after DNMTi treatment in Rh30 and RD

23 cells show changes in expression of the RASSF family (RASSF1,4,5, and 6), YAP1, myogenic

24 differentiation genes (MYOG, MYF5, and MYF6) and FGFR4. Fold change (average of both cell

1 lines) compared to the DMSO control is shown. (C) qRT-PCR validates the increase in expression

2 of RASSF1, RASSF4, RASSF5, and RASSF6 in a panel of RMS cell lines. (D) Immunoblots in

3 Rh30 aRMS cells (left) and RD eRMS cells (right) show an increase in RASSF1 and RASSF5

4 protein expression and a decrease in RASSF4 protein expression after DNMTi treatment. (E) MS-

5 PCR for the promoters of RASSF1 and RASSF5 demonstrates a demethylation of these loci after

6 DNMTi treatment in Rh30 aRMS cells (top) and RD eRMS cells (bottom). (F) Beta Values from

7 the EPIC methylation array for all RASSF4 probes that are significantly changed demonstrates a

8 demethylation of these loci after DNMTi treatment in Rh30 aRMS cells (top) and RD eRMS cells

9 (bottom).*, P<0.05; **, P<0.01; ***, P<0.001; and ****, P<0.0001.

10 Figure 3. DNMTi treatment inhibits YAP1 via a Hippo-dependent mechanism.

11 (A) LATS activity measured by the LATS activity luciferase assay doubles after DNMTi treatment

12 in Rh30 aRMS (left) and RD eRMS (right) cells. Transient LATS1 expression was used as an

13 assay positive control. (B) Inactive P-YAP1 increases by immunoblot in Rh30 aRMS cell line after

14 DNMTi treatment. Active P-Y416 SRC family kinase and total YES1 also decrease after DNMTi

15 treatment. (C) Overexpression of constitutively active YAPS127A partially rescues the growth

16 inhibition after DNMTi treatment in Rh30 cells (light purple). (D) Immunoblot validation of YAP1

17 overexpression in YAPS127A expressing Rh30 cells. Bands are from the same blot but have

18 been rearranged into this order. ****, P<0.0001.

19 Figure 4. YES1 regulates YAP1 activity in a Hippo-independent manner.

20 (A) Proximity ligation assays performed with YES1 and YAP1 antibodies demonstrates YES1 and

21 YAP1 are in close proximity (yellow spots) in Rh30 aRMS cells. Nuclear spots are quantified.

22 Each color represents a biological replicate. (B) Cell fractionation after lentivirally expressed

23 YES1 shRNAs shows an enrichment of YAP1 in the cytoplasmic fraction in Rh30 aRMS cells.

24 NT= non-targeting shRNA. Vimentin is a loading control, Histone H3 and MEK1/2 are fractionation

1 controls. (C) qRT-PCR after YES1 suppression in Rh30 aRMS cells shows a decrease in

2 expression of YES1 and YAP1 target genes CTGF and CYR61. qRT-PCR performed 48 hours

3 after end of puromycin selection. (D) qRT-PCR following treatment with 0.5µM Dasatinib (Das)

4 for 48 hours also shows a decrease in CTGF and CYR61 expression in a panel of RMS cell lines.

5 (E) IF for YAP1 with DAPI counterstain demonstrates a shift of YAP1 to the cytoplasm after 3

6 days of 0.5µM Das treatment (top right) in Rh30 aRMS cells. Expression of a Dasatinib resistant

7 YES1 (YEST348I) can prevents a shift in YAP1 after Das treatment (bottom right). Images at

8 200X. (F) Immunoblot validation of YES1 overexpression in YEST348I expressing Rh30 aRMS

9 cells. ***, P<0.001; and ****, P<0.0001.

10 Figure 5. Combination treatment ablates RMS cell growth in vitro.

11 (A) IncuCyte growth curves of Rh30 aRMS cells (left) and RD eRMS cells (right) treated with

12 DNMTi alone (0.5µM), Dasatinib alone (0.5µM), or combination at 0.25µM and 0.5µM. (B)

13 Combination treatment causes an increase in cleaved PARP in Rh30 aRMS cells as measure by

14 immunoblot. (C) Immunoblots for P-AKT and AKT and NF-kB p65 in a panel of RMS cell lines

15 after combination treatment (0.5µM). *, P<0.05; **, P<0.01; ***, P<0.001; and ****, P<0.0001.

16 Figure 6. Combination treatment in orthotopic aRMS xenografts.

17 (A) Tumor growth over time by caliper measurements in Rh30 aRMS orthotopic xenografts. N= 5

18 mice per group. Treatment started at Day 10. (B) Tumor weight at end point. (C) qRT-PCR for

19 MYF6 demonstrates an increase in MYF6 expression after DNMTi and combination treatment in

20 Rh30 tumors. (D) Model of YAP1 signaling in RMS cells. YES1 promotes nuclear YAP1

21 localization and YES1 can be inhibited by Dasatinib. RASSF1 and RASSF5 drive inactive P-YAP

22 through Hippo signaling while RASSF4 inhibits P-YAP. DNMTi treatment can activate RASSF1

23 and RASSF5 and inhibit RASSSF4 to inactivate YAP1 signaling. YAP1 functions as a

1 transcriptional co-activator driving RMS growth and survival. *, P<0.05; **, P<0.01; ***, P<0.001;

2 and ****, P<0.0001.

Targeting Hippo-dependent and Hippo-independent YAP1 signaling for the treatment of childhood rhabdomyosarcoma

Katherine K Slemmons, Choh Yeung, Joshua T Baumgart, et al.

Cancer Res Published OnlineFirst April 30, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-3853

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