Requirement for YAP1 signaling in myxoid liposarcoma

Abstract Myxoid liposarcomas (MLS), malignant tumors of adipocyte origin, are driven by the FUS‐DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS‐DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. Here we show, using an unbiased functional genomic approach, that FUS‐DDIT3‐expressing mesenchymal stem cells and MLS cell lines are dependent on YAP1, a transcriptional co‐activator and central effector of the Hippo pathway involved in tissue growth and tumorigenesis, and that increased YAP1 activity is a hallmark of human MLS. Mechanistically, FUS‐DDIT3 promotes YAP1 expression, nuclear localization, and transcriptional activity and physically associates with YAP1 in the nucleus of MLS cells. Pharmacologic inhibition of YAP1 activity impairs the growth of MLS cells in vitro and in vivo. These findings identify overactive YAP1 signaling as unifying feature of MLS development that could represent a novel target for therapeutic intervention.

Thank you for the submission of your manuscript to EMBO Molecular Medicine, and my apologies for the delay in getting back to you, which is due to the late review from one referee. We have now heard back from the three referees who were asked to evaluate your manuscript. As you will see from the reports below, they are overall positive and support publication of the article in EMBO Molecular Medicine pending appropriate revisions. They however highlight the need to further strengthen the study through minor additional experiments, and more thorough discussion and clarifications. Mouse experiments (mentioned by reviewer #3) will not be required for acceptance of the manuscript. EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.
EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. Please also contact us as soon as possible if similar work is published elsewhere. If other work is published, we may not be able to extend the revision period beyond three months.
Please read below for important editorial formatting and consult our author's guidelines for proper formatting of your revised article for EMBO Molecular Medicine.
I look forward to receiving your revised manuscript. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author): This is an excellent paper based on robust methodology. Research has been undertaken using variety of approaches that seems to be coherent. Non ethical issues should be raised.

Referee #1 (Remarks for Author):
This is an excellent paper demonstrating the role of YAP in myxoid liposarcoma molecular oncogenesis. As authors suggest that YAP involvement may be limited to MLPS they may briefly comment how should be interpreted YAP up-regulation in approximately one third of dedifferentiated liposarcoma.
Referee #2 (Remarks for Author): Trautman et al., explore genes required for survival of myxoid liposarcoma cells, which are transformed by the FUS-DDIT3 transcription factor fusion. Using an shRNA screen, they identify YAP as the top target and then perform a range of in vitro, patient sample and transcriptional studies that go some way to validating YAP as an important downstream gene relevant for myxoid liposarcoma. They also provide hints at mechanism by showing that YAP and FUS-DDIT3 can form a physical complex. In order to fully validate that these proteins operate together to regulate transcription it would require substantially more work, but I do not deem that such work is necessary for the current manuscript.
In summary, the study is generally well performed and has interesting findings that will spur future studies. I have some minor comments aimed at improving the manuscript.
Minor comments -it would be valuable for readers to know more about the results of the screen, e.g. by providing a full gene list. This would enable readers to, for example, see where other cancer genes and Hippo pathway genes other than YAP scored in the screen.
The legend for Figure 2 is inaccurate: "Increased YAP1 activity in FUS-DDIT3-expressing mesenchymal stem cells, MLS cell lines, and MLS patient samples." YAP expression and subcellular localization, but not activity, is assessed. Nuclear YAP/Yorkie does not always correspond with its activity.
The co-IPs in Figure 4E are missing an important control. The V5 pulldown should be performed in cells expressing FUS-DDIT3 but not V5-YAP and then the V5 pulldown performed to determine whether FUS-DDITS3 can be non-specifically precipitated by anti-V5.
Care should be taken when interpreting the verteporphin studies (cell line and chicken CAM) and this should be reflected in the manuscript as the drug has cytotoxic effects and in some settings these have been shown to be independent of YAP, TAZ and TEADs.
Referee #3 (Comments on Novelty/Model System for Author): The paper is extremely well written. The experiments are well controlled and well presented. Findings from this paper establish a new target for therapeutic intervention in a rare, aggressive form of cancer.

Referee #3 (Remarks for Author):
Trautmann et al identify YAP1, a central component of the Hippo pathway, as an important oncogenic effector in mesenchymal cells expressing the FUS:DDIT3 fusion oncogene. They show increased YAP1 activity in myxoid liposarcoma tissue and propose that YAP1 and FUS:DDIT3 interact to coordinate a gene expression program that promotes myxoid liposarcoma malignancy. Finally, they demonstrate that Verteporfin, which inhibits YAP1, inhibits myxoid liposarcoma growth in cell lines and chicken CAM.
The paper is extremely well written. The experiments are well controlled and well presented. Findings from this paper establish a new target for therapeutic intervention in a rare, aggressive form of cancer.
The paper does not address how YAP1 and FUS:DDIT3 interact to promote myxoid liposarcoma malignancy. Also, it would be nice to see mouse data on the potential efficacy of Verteporfin against myxoid liposarcoma. The Verteporfin doses used to achieve anti-tumor effects are fairly high; it may be difficult to achieve effective levels in vivo.
I expect that the authors will direct their attention towards mechanism of actions and further preclinical validation in future studies.
I recommend acceptance of the paper. We thank the Reviewers and the Editor for their insightful and constructive comments, which have substantially improved this work. Please find enclosed a revised manuscript that has been modified in accordance with their recommendations. Our specific responses to the Reviewers' comments are detailed individually below.

Referee #1 (Comments on Novelty/Model System for Author):
This is an excellent paper based on robust methodology. Research has been undertaken using variety of approaches that seems to be coherent. Non ethical issues should be raised.
We are grateful for these favorable comments.

Referee #1 (Remarks for Author):
This is an excellent paper demonstrating the role of YAP in myxoid liposarcoma molecular oncogenesis. As authors suggest that YAP involvement may be limited to MLPS they may briefly comment how should be interpreted YAP up-regulation in approximately one third of dedifferentiated liposarcoma.
We thank the Reviewer for this helpful question. Indeed, the wording of the abstract implied that YAP1 upregulation represents a unique feature of myxoid liposarcoma (MLS), and the Reviewer is right in pointing out that a subset of dedifferentiated liposarcoma (DDLS) also express YAP1 ( Figure 2E and F), albeit to a much lesser extent. This finding is in agreement with data by

Referee #2 (Remarks for Author):
Trautman et al., explore genes required for survival of myxoid liposarcoma cells, which are transformed by the FUS-DDIT3 transcription factor fusion. Using a shRNA screen, they identify YAP as the top target and then perform a range of in vitro, patient sample and transcriptional studies that go some way to validating YAP as an important downstream gene relevant for myxoid liposarcoma. They also provide hints at mechanism by showing that YAP and FUS-DDIT3 can form a physical complex. In order to fully validate that these proteins operate together to regulate transcription it would require substantially more work, but I do not deem that such work is necessary for the current manuscript. In summary, the study is generally well performed and has interesting findings that will spur future studies. I have some minor comments aimed at improving the manuscript.
We are delighted that the Reviewer found this a well-performed study that has yielded interesting results and will stimulate future investigations.
Minor comments -it would be valuable for readers to know more about the results of the screen, e.g. by providing a full gene list. This would enable readers to, for example, see where other cancer genes and Hippo pathway genes other than YAP scored in the screen.
We agree and have included the results of the RNA interference (RNAi) screen conducted in human mesenchymal stem cells in the revised manuscript. Specifically, we now provide the full gene list generated by RNAi Gene Enrichment Ranking, which was employed to rank genes with respect to FUS-DDIT3-selective essentiality, as Appendix Table S1 (page 4; lines 115-116).
The legend for Figure 2 is inaccurate: "Increased YAP1 activity in FUS-DDIT3-expressing mesenchymal stem cells, MLS cell lines, and MLS patient samples." YAP expression and subcellular localization, but not activity, is assessed. Nuclear YAP/Yorkie does not always correspond with its activity.
We agree and have amended the legend to Figure 2 (page 24; line 772).
The co-IPs in Figure 4E are missing an important control. The V5 pulldown should be performed in cells expressing FUS-DDIT3 but not V5-YAP and then the V5 pulldown performed to determine whether FUS-DDITS3 can be non-specifically precipitated by anti-V5.
Thank you for this helpful suggestion. We have performed this experiment and found that FUS-DDIT3 was not precipitated by the anti-V5 antibody ( Figure 4E).
Care should be taken when interpreting the verteporfin studies (cell line and chicken CAM) and this should be reflected in the manuscript as the drug has cytotoxic effects and in some settings these have been shown to be independent of YAP, TAZ and TEADs.
We completely agree with the Reviewer that the specificity of verteporfin is limited. Verteporfin was initially described as a photosensitizer that is used in photodynamic therapy of neovascular macular degeneration, where it is activated by laser light to generate reactive oxygen species . Zhang et al. described inhibitory effects of verteporfin in different colon cancer models that were not related to blockade of YAP1 or TAZ activity but due to impaired clearance of high-molecular-weight protein aggregates (Sci Signal 8:ra98, 2015). Thus, it is likely that verteporfin can affect cancer cells through several mechanisms. However, given the concordance between our pharmacologic data and the results obtained by various genetic approaches, we are confident that the effects of verteporfin in MLS cells are primarily due to YAP1-TEAD complex inhibition. Nonetheless, we agree with the Reviewer that a more nuanced discussion of the drug's properties is warranted, and we have amended the Discussion section accordingly (page 9; lines 276-281).

Referee #3 (Comments on Novelty/Model System for Author):
The paper is extremely well written. The experiments are well controlled and well presented. Findings from this paper establish a new target for therapeutic intervention in a rare, aggressive form of cancer.
Thank you for these positive and encouraging comments. We share the Referee's view that YAP1 represents a novel therapeutic target that warrants further study to improve outcomes for patients with MLS.

Referee #3 (Remarks for Author):
Trautmann et al identify YAP1, a central component of the Hippo pathway, as an important oncogenic effector in mesenchymal cells expressing the FUS:DDIT3 fusion oncogene. They show increased YAP1 activity in myxoid liposarcoma tissue and propose that YAP1 and FUS:DDIT3 interact to coordinate a gene expression program that promotes myxoid liposarcoma malignancy. Finally, they demonstrate that Verteporfin, which inhibits YAP1, inhibits myxoid liposarcoma growth in cell lines and chicken CAM.
The paper is extremely well written. The experiments are well controlled and well presented. Findings from this paper establish a new target for therapeutic intervention in a rare, aggressive form of cancer.
The paper does not address how YAP1 and FUS:DDIT3 interact to promote myxoid liposarcoma malignancy. Also, it would be nice to see mouse data on the potential efficacy of Verteporfin against myxoid liposarcoma. The Verteporfin doses used to achieve anti-tumor effects are fairly high; it may be difficult to achieve effective levels in vivo. I expect that the authors will direct their attention towards mechanism of actions and further preclinical validation in future studies. I recommend acceptance of the paper.
We are grateful for these favorable comments. As pointed out by the Reviewer, the mechanistic details of the interaction between FUS-DDIT3 and YAP1 need to be deciphered in future studies. Our current hypothesis is that FUS-DDIT3 and YAP1 interact in chromatin-associated multi-protein complexes whose analysis will require extensive proteomic experimentation.
We are aware of the limitations of the avian chorioallantoic membrane (CAM) model as an in vivo tool. However, the only patient-derived xenograft model reported thus far (Qi et al. Oncotarget 8:54320-30, 2017) is not available to us, and previous attempts to establish and propagate MLS cell line-based xenotransplants have, to our best knowledge, not been successful. We will continue our efforts to establish a mammalian in vivo system for future experiments. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees. As you will see the reviewers are now supportive, and I am pleased to inform you that we will be able to accept your manuscript pending minor editorial amendments.
Referee #2 (Remarks for Author): The authors have responded to all of the reviewer queries, which were on the minor end of the scale.
Referee #3 (Remarks for Author): The reviewers' concerns have been addressed, and I recommend acceptance of the paper. Authors made the requested changes.

USEFUL LINKS FOR COMPLETING THIS FORM
! ! ! ! " common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; " are tests one-sided or two-sided? " are there adjustments for multiple comparisons? " exact statistical test results, e.g., P values = x but not P values < x; " definition of 'center values' as median or average; " definition of error bars as s.d. or s.e.m.
1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria preestablished?
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe.
For animal studies, include a statement about randomization even if no randomization was used.
4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. All IHC studies were scored by pathologists (W.H., S.H., E.W.) blinded to the specific liposarcoma subtype and outcome data. The semiquantitative IHC score cutpoint (positive = semiquantitative score greater than 2) was prespecified without prior analyses. The staining intensity of the positive control (hepatocellular carcinoma) was defined as strong. Negative control stainings using an appropriate IgG subtype (DCS) were included.
NA -no animal experiment/studies were performed 1. Data the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes # (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. Yes -Variance was assessed by F test for data analyzed by t test in GraphPad Prism. The variance of data analyzed by two-way ANOVA was not assessed by GraphPad Prism. Yes see Appendix Table S2 see Material & Method section (Cell culture; page 11) NA -no animal experiment/studies were performed NA -no animal experiment/studies were performed NA -no animal experiment/studies were performed G-Dual use research of concern F-Data Accessibility