Glutamine-Utilizing Transaminases Are a Metabolic Vulnerability of TAZ/YAP-Activated Cancer Cells

EMBO reports - Peer Review Process File

Glutamine-utilizing transaminases are a metabolic vulnerability of TAZ/YAP-activated cancer cells

Chih-Sheng Yang, Eleni Stampouloglou, Nathan M Kinston, Liye Zhang, Stefano Monti, Xaralabos Varelas

Review timeline: Submission date: 26 October 2016 Editorial Decision: 24 November 2016 Resubmission: 17 January 2018 Editorial Decision: 6 February 2018 Revision received: 19 March 2018 Accepted: 23 March 2018

Editor: Achim Breiling

Transaction Report:

(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision 24 November 2018

Thank you for the submission of your research manuscript to EMBO reports. We have now received the full set of referee reports that are copied below.

I am sorry to say that the decision on your manuscript is not a positive one. As you will see, referees #1 and #2 do not provide strong support for the publication of your study in EMBO reports. Both referees, in particular referee #1, have significant concerns, and point out that your conclusions are not sufficiently supported by the data. Referee #3 is more positive,but also s/he has many comments that would need to be addressed.

Given these comments, the amount of work required to address them, and the fact that EMBO reports can only invite revision of papers that receive enthusiastic support from the referees upon initial assessment, I am sorry to say that we cannot offer to publish your manuscript.

I am sorry to have to disappoint you this time. I nevertheless hope, that the referee comments will be helpful in your continued work in this area, and I thank you once more for your interest in our journal.

REFEREE REPORTS ------Referee #1:

I have evaluated the MS and found many flaws that I would find very difficult to address in a rebuttal. Authors interrogated transcriptional profiles of MDA231 cells for signatures of specific metabolic cascades, and found that TAZ and YAP inactivation causes downregulation of 13 different cellular metabolic branches.

EMBO reports - Peer Review Process File

1) This is already the first problem. Very obviously, deficiency of YAP and TAZ from cells so relying on these factors has catastrophic consequences. This is raising a specificity and primary vs. secondary etc issue. Are we looking at cells that are on the way of growth arresting or dying , starting to shut down a substantial fraction of cellular metabolic and non-metabolic cascades? Are these genes directly regulated by YAP/TAZ or only secondarily, due for example to inhibition of E2F or other pathways downstream to YAP/TAZ? 2) The authors decided to focus on glutamine dependency and found that out of 10 cell lines tested 2 are addicted to exogenous glutamine, MDAMB231 and HCC38. What is the generality and significance of these results? 3) Are all the other non-glutamine addicted cells also insensitive to YAP or TAZ inhibition? This would be very surprising. 4) Is the result they obtain in just 2 cell lines dependent on the culture conditions? What if they adopt soft agar or other suspension cultures? 5) They attempt to correlate this with the fact that the two sensitive cell lines display higher TAZ or YAP Levels. However, these correlations do not demonstrate any functional hint and are compared to nothing else (how many transcription factors or oncogenes would show the same association?) making this difficult to evaluate. 6) Next they show that TAZ/YAP deficiency protected MDAMB231 against cell death and prevented the decline of the cell population caused by glutamine deprivation. This is a result at odd with several other reports in which YAP and TAZ inactivation is incompatible with cell growth of many cell types, both in vitro and in vivo (development, organ growth) that is under conditions in which glutamine was not deprived or in limited amounts. How is it possible that YAP/TAZ siRNAs do not cause any growth arrest in these cells? This casts serious doubts on the efficiency and specificity of the reagents they are using. Moreover, in cancer settings, endogenous tumors in mouse models are dependent on YAP and TAZ, an effect obviously unrelated to any in vitro + cell line specific + siRNA-dependent + culture manipulation-dependent they observe here. And the same is true for a wealth of literature of cells lines with manipulated YAP and TAZ levels, whereby inactivation of either YAP, TAZ or both is blocking tumor growth in vivo (xenog.). I think this sufficiently suggests that they are looking at a very peculiar idiosyncrasy of their experimental set up. But this is far from being informative for the readers of this journal. 7) Also puzzling is why, according to their MDAMB231 model, parental cells are unable to rewire their metabolism to cope glut shortage but not YAP-depleted cells. The simplest alternative is that Glut deficiency is not correlated or linked directly to YAP and TAZ but, merely, to the growth rate of different cell types (in turn affected by the culture conditions etc). Cells with a partial YAP and TAZ depletion would be alive but growth impaired, making the glut input much less relevant (back to my point 1). 8) Finally, the authors never provide evidence for effective rewiring of metabolism of their cells upon manipulation of YAP/TAZ activity. They only show what happens by depleting glutamine from the medium, and by adding back AKG which anyway can be rapidly redirected in several metabolic pathways through forward and reverse reactions in the TCA cycle. This might simply indicate that some cells are better than others not at rewiring glutamine metabolism, but at enhancing the usage of parallel carbon sources to proliferate. Also on this point, gene-expression data on regulation of metabolic enzymes are only based on microarray analysis (which does not automatically mirrors effective gene regulation), correlations, and on a very minor regulation of GOT and PSAT1 genes, which might be irrelevant to effectively rewire metabolism in cells.

------Referee #2:

This is a potentially significant study that links TAZ/YAP-mediated growth of breast cancer cells to glutamine metabolism. The major strength of the study is its novelty and timeliness, given the surge of interest in understanding how TAZ/YAP and metabolism contribute to cancer and their potential as therapeutic targets. Some aspects of this study, however, need to be strengthened and the conclusions tempered somewhat.

1. Figure 1 needs clarification. The authors correlate TAZ/YAP expression with glutamine dependence but make no mention of TAZ/YAP activity as they do in Fig. 4. Do the data points shown in Fig. 1D represent individual cell lines and are these data culled from 1B and 1C? 2. The authors group TAZ and YAP in their experiments but the existing evidence indicates that

they may not have overlapping functions in breast cancer and that TAZ may be associated more with clinical outcomes (reviewed in Cancer Cell 2016, 29:783). Also, TAZ/YAP activity has been shown to be important for the function of breast cancer stem cells. Are the observed effects on growth independent of stem cell function? These points warrant discussion if not a few experiments. For example, individual knock-down of TAZ and YAP would be helpful. 3. The quality of the photomicrographs in Fig. 2A is poor and these data should be quantified. 4. A major conclusion of the study is that TAZ/YAP 'induce' GOT1 and PSAT1 expression. Based on the data provided (Fig. 4 C, D), the verb 'induce' is too strong. The immunoblot shown on 4C would benefit from densitometry. Given that this is an important figure, a rescue experiment for the siRNA would be helpful. 5. The figure legend to 4D does not make sense. 6. The conclusion that AOA suppresses growth in a TAZ/YAP-dependent manner seems a bit of a stretch, especially for MDA-231 cells (Fig. 4F).

------Referee #3:

This appropriately formatted paper is novel, physiologically relevant, has broad biological significance is important to the field of cancer metabolism. While the conclusions are well supported by the data, some work remains to elucidate a few remaining details.

Summary Overview: Metabolic reprogramming of cancer metabolism, including reprogrammed glutamine metabolism, is viewed as a therapeutic target. However, there is a limited understanding of which tumors will be sensitive to different types of anti-metabolism theories. Understanding how driver oncogenes link to metabolism will help prioritize which tumors may respond to anti-metabolism therapies. Here, Yang et al. show a novel connection between TAZ/YAP oncogenes and glutamine catabolism in breast cancer cells, in contrast with a paper which shows TAZ/YAP drive glutamine synthesis in a zebrafish liver model. While the paper currently has some outstanding questions that need addressed, the YAP/TAZ link to glutamine catabolism is of interest with further experiments and revisions. Major claims: These claims are novel and the authors do not make claims that exceed their data, Utilizing publicly available YAP/TAZ knockdown microarray dataset from a paper from a different lab that previously linked YAP/TAZ to aerobic glycolysis, another noted change in cancer metabolism, the authors found downregulation of YAP/TAZ downregulates amino acid metabolism. In figure 1, the authors show knockdown of YAP/TAZ causes downregulation of amino acid metabolism. High YAP/TAZ cells show increased glutamine dependence for growth. In figure 2, the authors show that knockdown of YAP/TAZ decreases cell deaths following glutamine depletion. Consistent with several other papers in the literature focusing on glutamine metabolism in breast cancer, in figure 3 the authors use re-addition of glutamine metabolites to show that the major metabolic fate of glutamine is contribution to the TCA cycle. Glutamate can be converted to a-KG be either amino acid creating aminotransferases or glutamate dehydrogenase. In figure 4 the authors use TCGA RNA-seq data to show aminotransferases GOT1, PSAT1, GPT2 and GOT2 positively correlate with a YAP/TAZ activity score while GLUD1/2 in negatively correlated with YAP/TAZ activity score. The authors then showed ~40% decrease in GOT1 and PSAT1 mRNA and a larger decrease in mRNA in YAP/TAZ knockdown cells. Finally the authors show that high YAP/TAZ cells have greater sensitivity to inhibition of aminotransferases, and that knockdown of YAP/TAZ decreases the sensitivity to aminotransferase inhibitors.

Major concerns: 1) This paper lacks gain of function data to show YAP/TAZ is sufficient to induce glutamine dependence. Does ectopically expressing YAP/TAZ in YAP/TAZ low cells induce glutamine dependence in BT474 and HMT-3522 S1 cells? 2) The paper lacks mechanism for the regulation of the glutamine metabolism genes. ChIP-Seq data is publicly available for YAP/TAZ in MDA-MB-231 cells. Zanconato et. al. Nat Cell Biol. 2015 Sep;17(9):1218-27. Are there binding sites regulating glutamine metabolism genes? 3) The interest of this paper would be greatly increased if the authors managed to link YAP/TAZ observation to the larger glutamine dependence literature. Zanconato et al. Nat Cell Biol. 2015 Sep;17(9):1218-27. previously showed in MDA-MB-231 that YAP/TAZ directly regulates MYC and that MYC re-expression can partially rescue growth following YAP/TAZ knockdown. MYC has

been shown to induce glutamine dependence in a number of contexts, and has been shown to induce dependence on glutamine downstream of other pathways such as mTOR (Csibi Curr Biol. 2014 Oct 6;24(19):2274-80). Korangath Clin Cancer Res. 2015 Jul 15;21(14):3263-73 has previously shown in breast cancer that knockdown of MYC can decrease sensitivity of breast cancers to aminooxyacetate. Determining if MYC re-expression was sufficient to restore glutamine dependence and aminooxyacetate following YAP/TAZ knockdown would help fit this paper into the larger body of glutamine metabolism media. 4) Further efforts should be made to connect the aminooxyacetate data to the glutamine free data. A) Does AOA induce cell death? B) Is AOA rescued by alpha-ketoglutarate? 5) The authors don't show the effect of TAZ/YAP knockdown on growth. Coloff et al. Cell Metab. 2016 May 10;23(5):867-80. Showed using confluent cells that quiescent mammary epithelial cells decrease dependence on amino transferase. Is the TAZ/YAP protection against glutamine depletion due to decreased growth or alteration of mTOR activity? Can enforced TAZ/YAP expression maintain high aminotransferases in confluent cells

Minor concerns

1) There is a lack of consistency in the logic linking YAP/TAZ to expression of glutamine metabolism genes. While the authors begin by using microarray data from another paper to show that the amino acid metabolism genes are downregulated after YAP/TAZ deletion (Fig 1A), in Table EV1, only GPT and GPT2 are part of the core pathway in the flux of glutamine to a-KG. GPT and GPT2 are ignored largely throughout the rest of the paper. The paper would be strengthened by further validation of the microarray by measuring the effect of YAP/TAZ knockdown on mRNA expression of glutamine metabolism (SLC1A5, SLC38A1, SLC38A2, GLS, GPT, GPT2, GOT2, SLC25A22) genes beyond the examination PSAT1 and GOT1 shown in figure 4D. The authors show upregulation of GLUD1/2 at protein but not mRNA level in figure 4D. 2) The authors duplicate a lot of work already done in breast cancer, and fail to cite the previous work focusing on glutamine dependence in triple negative breast cancer (Timmerman et al. Cancer Cell. 2013 Oct 14;24(4):450-65, Gross et al. Mol Cancer Ther. 2014 Apr;13(4):890-901.) The authors also fail to link their data to previous work with aminooxyacetate in breast cancer (Korangath Clin Cancer Res. 2015 Jul 15;21(14):3263-73). The authors also fail to address the work of Coloff et al. Cell Metab. 2016 May 10;23(5):867-80 focusing on GLUD vs aminotransferase in mammary tissue and breast cancer. 3) Korangath Clin Cancer Res. 2015 Jul 15;21(14):3263-73 have shown previously that consistent with the role of GOT1/GOT2 synthesis of aspartate through the TCA cycle, that media aspartate can alter the sensitivity of cells to aminooxyacetate. The most sensitive cell line in the study (MDA-MB- 231) is the only cell line grown in media without aspartate as RPMI and DMEM/F12 typically have aspartate, while the MDA-MB-231 DMEM media does not. Does aspartate rescue the MDA-MB- 231 AOA phenotype? (Acknowledging that NEAA acids were unable to rescue glutamine free growth in figure 3). 4) As aminooxyacetate is a very broad drug that targets dozens of aminotransferases, this paper would be strengthened by knockdown of separate aminotransferase to determine which enzymes are the key aminotrasferases. While the authors discuss PSAT1 in MDA-MB-231 cells, a body of literature (for example Possemato Nature. 2011 Aug 18;476(7360):346-50) suggests that serine synthesis is limited in that cell line. 5) Due to the broad effects of aminooxyacetate, a more direct measure of the sensitivity of high/low YAP/TAZ to the inhibition of glutamine to a-KGA may be the inhibition of glutaminase using 968/BPTES/CB-839.

Resubmission 17 January 2018

Referee #1:

RESPONSE: We thank the reviewer for their comments. To address the first comment, we performed additional experiments to show a direct relationship between TAZ/YAP and glutamine metabolism regulation. The ChIP experiments we show in revised Fig 3 indicate that TAZ and the TEAD transcription factors are recruited to an enhancer for the GOT1 gene. We also show that TAZ/YAP knockdown reduces GOT1 gene expression and protein levels, and that ectopic TAZ expression is sufficient to sustain high levels of GOT1 expression. Further, our results in revised Fig 4 show that blockade of glutamine-utilizing transaminases slows the growth of breast cancer cells expressing high levels of TAZ/YAP, placing glutamine metabolism upstream to cancer cell growth. Therefore, our data strongly suggest that the GOT1 transaminase is a direct TAZ/YAP target, and the reduction of glutamine dependence following TAZ/YAP knockdown is not merely a secondary effect.

2) The authors decided to focus on glutamine dependency and found that out of 10 cell lines tested 2 are addicted to exogenous glutamine, MDAMB231 and HCC38. What is the generality and significance of these results?

RESPONSE: Our conclusion from the experiments shown in Fig 1 is that TAZ/YAP levels strongly correlate with glutamine dependency. This is based on our own experimental analysis of 10 mammary/breast cancer cells line, not just results from MDA-MB-231 and HCC38. Glutamine dependency is not an all or none phenotype and, as indicated in our manuscript, cells expressing intermediate levels of TAZ/YAP show less growth suppression following the removal of glutamine than cells with high levels of TAZ/YAP. Further, our analysis of gene expression data from human breast cancers available from the TCGA (1088 breast cancer biopsies) show a significant correlation between TAZ/YAP activity and the expression of the transaminases GOT1 and PSAT1 (revised Fig 3D). These data suggest that the relationship between YAP/TAZ and glutamine dependency is general and not specific to tested cell lines.

3) Are all the other non-glutamine addicted cells also insensitive to YAP or TAZ inhibition? This would be very surprising.

RESPONSE: Our data shown in Fig 1B-E show a correlation between TAZ/YAP levels and glutamine dependency, and indicates that non-glutamine addicted cells express very low levels of TAZ/YAP. Knockdown of TAZ/YAP in cancer cells with “low levels” of TAZ/YAP had minimal effects on cell growth in glutamine deficient media. However, as shown in Fig 2A-B, we show that cancer cells expressing high TAZ/YAP cannot survive in glutamine free media, and knockdown of TAZ/YAP rescues this growth deficiency.

4) Is the result they obtain in just 2 cell lines dependent on the culture conditions? What if they adopt soft agar or other suspension cultures?

RESPONSE: We apologize for the misunderstand of our presented data. In our manuscript, we examined ten cell lines, not just two. The culture conditions of each of these cell lines vary and are specified in Table EV3, so we can conclude that our data is not dependent on culture conditions.

5) They attempt to correlate this with the fact that the two sensitive cell lines display higher TAZ or YAP Levels. However, these correlations do not demonstrate any functional hint and are compared to nothing else (how many transcription factors or oncogenes would show the same association?) making this difficult to evaluate.

RESPONSE: As mentioned in our responses above, the correlation we show is built from results using ten cell lines (not just two), which indicates a relationship between TAZ/YAP and glutamine dependency. We also show that this effect is not simply correlative, as we show that TAZ/YAP depletion affects glutamine dependency (Fig 2) and that ectopic TAZ expression is sufficient to

rescue the observed effects of TAZ/YAP depletion (Fig 2E). Further, we identify GOT1, an important regulator of glutamine metabolism, as a direct target of TAZ-TEAD (Fig 3G), highlighting a functionally relevant mechanism linking TAZ/YAP activity to glutamine dependence.

6) Next they show that TAZ/YAP deficiency protected MDAMB231 against cell death and prevented the decline of the cell population caused by glutamine deprivation. This is a result at odd with several other reports in which YAP and TAZ inactivation is incompatible with cell growth of many cell types, both in vitro and in vivo (development, organ growth) that is under conditions in which glutamine was not deprived or in limited amounts. How is it possible that YAP/TAZ siRNAs do not cause any growth arrest in these cells? This casts serious doubts on the efficiency and specificity of the reagents they are using.

Moreover, in cancer settings, endogenous tumors in mouse models are dependent on YAP and TAZ, an effect obviously unrelated to any in vitro + cell line specific + siRNA-dependent + culture manipulation-dependent they observe here. And the same is true for a wealth of literature of cells lines with manipulated YAP and TAZ levels, whereby inactivation of either YAP, TAZ or both is blocking tumor growth in vivo (xenog.). I think this sufficiently suggests that they are looking at a very peculiar idiosyncrasy of their experimental set up. But this is far from being informative for the readers of this journal.

RESPONSE: We apologize for not conveying our study design in a clearer manner. In the cells examined we did observe slowed down cell growth following YAP/TAZ depletion in complete medium. However, in Fig 2 we assayed how knockdown of YAP/TAZ affects MDA-MB-231 “cell survival” under “glutamine deprived” conditions, and found that parental MDA-MB-231 and HCC38 cells (with high TAZ/YAP) cannot survive in glutamine deprived conditions as indicated by a sharp drop of cell number. On the contrary, cell numbers are “maintained” under the same culture condition if YAP/TAZ or TAZ alone were depleted by siRNA. We would argue that our data provide evidence that YAP/TAZ control cell survival under a metabolically advert condition such as glutamine withdrawal or pharmacological inhibition of glutamine metabolism. These observations therefore indicate that elevated TAZ/YAP levels mediate important events that promotes the glutamine dependence of breast cancer cells.

While we appreciate the reviewers concern, we would like to point out that the statements made by the reviewer relating YAP/TAZ to cell growth are not true in all contexts. As the reviewer stated, there are many examples of YAP/TAZ directing cell proliferation and survival, but there also several cell populations that can grow in the absence of YAP/TAZ. A prime example of this is the developing mouse intestinal epithelium, which has been reported to develop normally (i.e. epithelial cell proliferation, survival and fate are normal) following deletion of both the YAP and TAZ genes (see Azzolin et al, Cell 2014). Therefore, the coupling of YAP/TAZ to cell growth may not be as straight forward as the reviewer assumes.

7) Also puzzling is why, according to their MDAMB231 model, parental cells are unable to rewire their metabolism to cope glut shortage but not YAP-depleted cells. The simplest alternative is that Glut deficiency is not correlated or linked directly to YAP and TAZ but, merely, to the growth rate of different cell types (in turn affected by the culture conditions etc). Cells with a partial YAP and TAZ depletion would be alive but growth impaired, making the glut input much less relevant (back to my point 1).

RESPONSE: This point is similar to that brought up in point #1 by this reviewer. Please see our reply above to point #1. In brief, we have shown glutamine metabolism is a prerequisite for TAZ/YAP-driven growth. Therefore, this metabolic pathway offers a novel vulnerability point for YAP/TAZ-driven cancer cells.

8) Finally, the authors never provide evidence for effective rewiring of metabolism of their cells upon manipulation of YAP/TAZ activity. They only show what happens by depleting glutamine from the medium, and by adding back AKG which anyway can be rapidly redirected in several metabolic pathways through forward and reverse reactions in the TCA cycle. This might simply indicate that some cells are better than others not at rewiring glutamine metabolism, but at enhancing the usage of parallel carbon sources to proliferate. Also on this point, gene-expression

data on regulation of metabolic enzymes are only based on microarray analysis (which does not automatically mirrors effective gene regulation), correlations, and on a very minor regulation of GOT and PSAT1 genes, which might be irrelevant to effectively rewire metabolism in cells.

RESPONSE: In addition to our analysis of microarray-based gene expression, which shows correlation between YAP/TAZ activity and transaminase expression in human breast cancers (Fig 3D), we have shown YAP/TAZ depletion using two different sets of siRNA inhibits the expression of the transaminases GOT1 and PSAT1, both at mRNA and protein levels (Fig 3E-F). We also show that an enhancer associated with GOT1 gene expression is directly bound by TAZ and the TEAD transcription factors using ChIP methods (Fig 3G). Further, we show that blockade of transaminases resulted in growth suppression in cells with high TAZ/YAP levels, and that TAZ/YAP depletion can reverse this growth suppression (Fig 4). Collectively, we would argue that our data strongly suggest a direct relationship between TAZ/YAP and transaminase activity, and that this relationship offers a novel vulnerably point for breast cancer cells with high YAP/TAZ activity.

------Referee #2:

RESPONSE: We thank the reviewer for these comments. In our revised manuscript, we show a strong correlation between the expression of four canonical TAZ/YAP target genes (CTGF, CYR61, ANKDR1, and EDN1) and glutamine dependence (Fig 1E), which further strengthens our conclusions. Also, the results shown in Fig 1D are from the same cell lines we used in 1B and C. We have clarified this in the revised text and figure legend.

2. The authors group TAZ and YAP in their experiments but the existing evidence indicates that they may not have overlapping functions in breast cancer and that TAZ may be associated more with clinical outcomes (reviewed in Cancer Cell 2016, 29:783). Also, TAZ/YAP activity has been shown to be important for the function of breast cancer stem cells. Are the observed effects on growth independent of stem cell function? These points warrant discussion if not a few experiments. For example, individual knock-down of TAZ and YAP would be helpful.

RESPONSE: We have performed the suggested experiments testing the effects of individual depletion of YAP and TAZ, comparing them to double TAZ/YAP knockdown. Consistent with prior reports that suggest a more important role for TAZ in breast cancer cells, we show that TAZ depletion, but not YAP depletion, can rescue the growth effects following glutamine depletion. However, TAZ depletion alone did not have as strong of an effect as TAZ/YAP depletion, indicating that YAP can function redundantly, at least under conditions of low TAZ. We have commented on the more important roles for TAZ in breast cancer in our revised manuscript.

3. The quality of the photomicrographs in Fig. 2A is poor and these data should be quantified.

RESPONSE: The figures we submitted were compressed to fit the size limitation. We have tried to increase the resolution for better clarity. Also, the data shown in Fig 2A is included in the quantitation in Fig 1C.

4. A major conclusion of the study is that TAZ/YAP 'induce' GOT1 and PSAT1 expression. Based on the data provided (Fig. 4 C, D), the verb 'induce' is too strong. The immunoblot shown on 4C would benefit from densitometry. Given that this is an important figure, a rescue experiment for the siRNA would be helpful.

RESPONSE: We have repeated the experiments in Fig 4C of the original manuscript using an independent siRNA that knocks down TAZ and YAP levels to a larger degree, leading to a more pronounce reduction of GOT1 and PSAT1 RNA and protein levels. As suggested, we have also included densitometry quantitation of these data (revised Fig 3E).

5. The figure legend to 4D does not make sense.

RESPONSE: We thank the reviewer for catching this error. We have edited the legend accordingly.

6. The conclusion that AOA suppresses growth in a TAZ/YAP-dependent manner seems a bit of a stretch, especially for MDA-231 cells (Fig. 4F).

RESPONSE: We have observed that YAP/TAZ knockdown significantly, albeit not complete, rescued AOA-mediated growth suppression in MDA-MB-231from independent experiments. This level of rescue is comparable to other factors implicated in mediating AOA-mediated effects (see Korangath et al. Clin Cancer Res, 2015). The relatively minor effects observed in MDA-MB-231 cells could be attributed to the complexity of the genetic background of this advanced cancer cells. To strengthen our conclusions, we have now performed an experiment where we ectopically expressed of a TAZ derivative that is insensitive to siRNA-mediated depletion in MDA-MB-231 cells, and found that expression of TAZ restored the growth suppression effects mediated by AOA (Fig 4D).

------Referee #3:

Major claims: These claims are novel and the authors do not make claims that exceed their data, Utilizing publicly available YAP/TAZ knockdown microarray dataset from a paper from a different lab that previously linked YAP/TAZ to aerobic glycolysis, another noted change in cancer metabolism, the authors found downregulation of YAP/TAZ downregulates amino acid metabolism. In figure 1, the authors show knockdown of YAP/TAZ causes downregulation of amino acid metabolism. High YAP/TAZ cells show increased glutamine dependence for growth. In figure 2, the authors show that knockdown of YAP/TAZ decreases cell deaths following glutamine depletion. Consistent with several other papers in the literature focusing on glutamine metabolism in breast cancer, in figure 3 the authors use re-addition of glutamine metabolites to show that the major metabolic fate of glutamine is contribution to the TCA cycle. Glutamate can be converted to a-KG be either amino acid creating aminotransferases or glutamate dehydrogenase. In figure 4 the authors use TCGA RNA-seq data to show aminotransferases GOT1, PSAT1, GPT2 and GOT2 positively correlate with a YAP/TAZ activity score while GLUD1/2 in negatively correlated with YAP/TAZ activity score. The authors then showed ~40% decrease in GOT1 and PSAT1 mRNA and a larger decrease in mRNA in YAP/TAZ knockdown cells. Finally the authors show that high YAP/TAZ cells have greater sensitivity to inhibition of aminotransferases, and that knockdown of YAP/TAZ decreases the sensitivity to aminotransferase inhibitors.

RESPONSE: We have attempted to generate stable cell lines ectopically expressing TAZ or YAP in a variety of mammary and breast cancer cell lines. We were unable to generate cells that ectopically expressed TAZ or YAP in any of the “low TAZ/YAP” cells, such as the BT474 or HMT-3522 S1 cells, as these cells consistently died within the first passages. We were therefore unable to perform the suggested experiments. Interestingly, these observations suggest that some mammary gland epithelial cells and breast cancer cells are unable to tolerate high levels of TAZ/YAP, which could be an interesting for another study. We were able to generate MDA-MB- 231 cells stably expressing ectopic mouse TAZ that is resistant to the human siRNA we used. We used this cell line to examine the effects of TAZ expression following TAZ/YAP knockdown (essentially rescue experiments). These experiments showed that ectopic mouse TAZ expression was sufficient to rescue all the observed effects of endogenous TAZ/YAP knockdown, including reversing the growth suppression effects following glutamine withdrawal (Fig 2E) and AOA treatment (Fig 4D), as well as reversing the loss of GOT1 expression (Fig 3I). These data indicate that TAZ expression is sufficient to mediate the observed effects.

2) The paper lacks mechanism for the regulation of the glutamine metabolism genes. ChIP-Seq data is publicly available for YAP/TAZ in MDA-MB-231 cells. Zanconato et. al. Nat Cell Biol. 2015 Sep;17(9):1218-27. Are there binding sites regulating glutamine metabolism genes?

RESPONSE: We thank the reviewer for raising this point. As suggested we have examined the available ChIP-seq data and using this data as a guide we have confirmed that TAZ and the TEAD transcription factors bind to an enhancer of the GOT1 gene. In addition, we have shown that TAZ expression, but not MYC expression, can restore GOT1 expression following TAZ/YAP knockdown. This new data suggests that TAZ/TEAD complexes promote GOT1 expression directly, adding important new mechanism to our manuscript (revised Fig 3G).

3) The interest of this paper would be greatly increased if the authors managed to link YAP/TAZ observation to the larger glutamine dependence literature. Zanconato et al. Nat Cell Biol. 2015 Sep;17(9):1218-27. previously showed in MDA-MB-231 that YAP/TAZ directly regulates MYC and that MYC re-expression can partially rescue growth following YAP/TAZ knockdown. MYC has been shown to induce glutamine dependence in a number of contexts, and has been shown to induce dependence on glutamine downstream of other pathways such as mTOR (Csibi Curr Biol. 2014 Oct 6;24(19):2274-80). Korangath Clin Cancer Res. 2015 Jul 15;21(14):3263-73 has previously shown in breast cancer that knockdown of MYC can decrease sensitivity of breast cancers to aminooxyacetate. Determining if MYC re-expression was sufficient to restore glutamine dependence and aminooxyacetate following YAP/TAZ knockdown would help fit this paper into the larger body of glutamine metabolism media.

RESPONSE: We have referenced these prior studies in our revised manuscript, and have performed new experiments comparing the effects of TAZ expression to those of MYC expression in MDA-MB-231 cells. Our data confirm the prior observations that MYC levels are reduced following TAZ/YAP depletion. Interestingly, our data show that the levels of ectopically expressed MYC (from a CMV promoter) are also reduced following TAZ/YAP depletion, suggesting that TAZ/YAP mediate MYC levels post-transcriptionally, possibly via mechanisms described in (Mori et al. Cell 2014). We added discussion of this point in the revised manuscript. While we cannot exclude the contributions of MYC to the observed phenotypes, our data indicate that TAZ expression, but not MYC expression, rescues the loss of GOT1 expression following TAZ/YAP knockdown (revised Fig 3H). Thus, our data argue that TAZ directly mediates the regulation of GOT1 levels.

4) Further efforts should be made to connect the aminooxyacetate data to the glutamine free data. A) Does AOA induce cell death? B) Is AOA rescued by alpha-ketoglutarate?

RESPONSE: We have performed the suggested experiments and found that AOA induces a growth arrest with accompanying cell death in MDA-MB-231 cells (new Fig 4C & D) and that

this phenotype can be partially rescued by TAZ/YAP knockdown. We further have found that the growth suppression phenotype induced by AOA can be partially rescued by the addition of AKG (Fig EV5).

5) The authors don't show the effect of TAZ/YAP knockdown on growth. Coloff et al. Cell Metab. 2016 May 10;23(5):867-80. Showed using confluent cells that quiescent mammary epithelial cells decrease dependence on amino transferase. Is the TAZ/YAP protection against glutamine depletion due to decreased growth or alteration of mTOR activity? Can enforced TAZ/YAP expression maintain high aminotransferases in confluent cells.

RESPONSE: The reviewer brings up an interesting point. Unfortunately, despite several attempts we have been unable to stably express high levels of TAZ or YAP in normal human mammary epithelial cells that exhibit low levels of TAZ/YAP (these cells undergo an interesting form of cell death), and therefore have been unable to perform the suggested experiments. However, we have added reference to the paper from Coloff et al. and discuss it in the context of our manuscript.

Minor concerns 1) There is a lack of consistency in the logic linking YAP/TAZ to expression of glutamine metabolism genes. While the authors begin by using microarray data from another paper to show that the amino acid metabolism genes are downregulated after YAP/TAZ deletion (Fig 1A), in Table EV1, only GPT and GPT2 are part of the core pathway in the flux of glutamine to a-KG. GPT and GPT2 are ignored largely throughout the rest of the paper. The paper would be strengthened by further validation of the microarray by measuring the effect of YAP/TAZ knockdown on mRNA expression of glutamine metabolism (SLC1A5, SLC38A1, SLC38A2, GLS, GPT, GPT2, GOT2, SLC25A22) genes beyond the examination PSAT1 and GOT1 shown in figure 4D. The authors show upregulation of GLUD1/2 at protein but not mRNA level in figure 4D.

RESPONSE: To address this comment we have included a new figure (Fig EV1) that shows the changes in the expression of the noted genes implicated in glutamine metabolism following TAZ/YAP knockdown in MDA-MB-231 cells.

2) The authors duplicate a lot of work already done in breast cancer, and fail to cite the previous work focusing on glutamine dependence in triple negative breast cancer (Timmerman et al. Cancer Cell. 2013 Oct 14;24(4):450-65, Gross et al. Mol Cancer Ther. 2014 Apr;13(4):890-901.) The authors also fail to link their data to previous work with aminooxyacetate in breast cancer (KorangathClin Cancer Res. 2015 Jul 15;21(14):3263-73). The authors also fail to address the work of Coloff et al. Cell Metab. 2016 May 10;23(5):867-80 focusing on GLUD vs aminotransferase in mammary tissue and breast cancer.

RESPONSE: We have revised our manuscript to better reference and discuss the indicated papers, as well as other recent papers related to our study.

3) Korangath Clin Cancer Res. 2015 Jul 15;21(14):3263-73 have shown previously that consistent with the role of GOT1/GOT2 synthesis of aspartate through the TCA cycle, that media aspartate can alter the sensitivity of cells to aminooxyacetate. The most sensitive cell line in the study (MDA-MB-231) is the only cell line grown in media without aspartate as RPMI and DMEM/F12 typically have aspartate, while the MDA-MB-231 DMEM media does not. Does aspartate rescue the MDA-MB-231 AOA phenotype? (Acknowledging that NEAA acids were unable to rescue glutamine free growth in figure 3).

RESPONSE: As pointed by the reviewer, our data shown in Fig 3 showed that the addition of a mixture of NEAA was unable to fully restore growth following glutamine withdrawal. We performed similar experiments specifically examining the effects of AKG or aspartate to the AOA response. As shown in Fig EV5 in our revised manuscript we find that both AKG and Asp are able to partially rescue the AOA growth effects, consistent with the points that the reviewer brings up. These data suggest an important role for the GOT transaminases in mediating MDA-MB-231 cell growth.

4) As aminooxyacetate is a very broad drug that targets dozens of aminotransferases, this paper

would be strengthened by knockdown of separate aminotransferase to determine which enzymes are the key aminotrasferases. While the authors discuss PSAT1 in MDA-MB-231 cells, a body of literature (for example Possemato Nature. 2011 Aug 18;476(7360):346-50) suggests that serine synthesis is limited in that cell line.

RESPONSE: As suggested by the reviewer we knocked down individual aminotransferase using siRNA, but we did not observe any significant effects on MDA-MB-231 viability following knockdown of GOT1 and/or PSAT1. As shown in the figure below (Reviewer Fig 1), we observed complex redundancy following individual and double knockdown of various aminotransferase that could explain why we did not observe cell viability differences. For example, we observed increased GPT expression following GOT1 and PSAT1 knockdown. These data suggest an interrelationship between the control of aminotransferase expression, and we hope the reviewer agrees that understanding this redundancy with respect to cell growth is beyond the scope of the current study.

5) Due to the broad effects of aminooxyacetate, a more direct measure of the sensitivity of high/low YAP/TAZ to the inhibition of glutamine to a-KGA may be the inhibition of glutaminase using 968/BPTES/CB-839.

RESPONSE: We have performed the suggested experiment using BPTES, and have observed that breast cancer cells expressing high levels of TAZ/YAP are also sensitive to glutaminase inhibition. We show that depletion of TAZ/YAP can reverse the effects of BPTES treatment. These data indicate that interfering with glutaminase activity is another potential mechanism for targeting cells with high TAZ/YAP activity. We have added discussion of these data in our revised manuscript.

Reviewer Fig 1. Redundancy in aminotransferase expression in MDA-MB-231 cells. MDAMB- 231 cells were transfected with control siRNA or siRNA targeting GOT1 and/or PSAT1 and the expression of the indicated genes was examined by RT-qPCR. The average relative expression levels for each gene is shown from three independent experiments +SE (*p<0.05; ***p<0.001).

2nd Editorial Decision 6 February 2018

Thank you for the re-submission of your research manuscript to EMBO reports. We have now received reports from the three referees that were asked to re-evaluate your study, which can be found at the end of this email. As you will see, all three referees now support the publication of your study in EMBO reports. However, referee #3 has a final concern we ask you to address in a final revised version of the manuscript.

Further, I also have the following editorial requests:

Most importantly, please provide individual files for EV figures, datasets and tables. For datasets and tables an excel file is fine, but please put a legend on the first tab of the file.

Present tables EV1 + EV4 are too long/wide to be displayed as a table. Please upload these as dataset (Dataset EV1 and Dataset EV2). Tables EV2 and EV3 would then be the new Tables EV1 and EV2. Please update all the call-outs in the manuscript text accordingly.

The title is currently too long. Please provide a simpler title with not more than 100 characters (including spaces).

Please provide the abstract written in present tense.

Regarding data quantification and statistics, can you please specify, where applicable, the number "n" for how many experiments were performed, the bars and error bars (e.g. SEM, SD) and the test used to calculate p-values in the respective figure legends. Please provide statistical testing where applicable. See: http://embor.embopress.org/authorguide#statisticalanalysis

We now strongly encourage the publication of original source data with the aim of making primary data more accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. If you would like to use this opportunity, please submit the source data (for example scans of entire gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key experiments together with the revised manuscript. Please include size markers for scans of entire gels, label the scans with figure and panel number, and send one PDF file per figure.

In addition I would need from you: - a short, two-sentence summary of the manuscript - two to three bullet points highlighting the key findings of your study - a schematic summary figure (in jpeg or tiff format with the exact width of 550 pixels and a height of about 400 pixels) that can be used as visual synopsis on our website.

Please also note that we now mandate that all corresponding authors list an ORCID digital identifier that is linked to their EMBO reports account!

I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision.

REFEREE REPORTS ------Referee #1: the MS has improved

------Referee #2:

The authors have responded adequately to the concerns I raised in the initial review.

------Referee #3:

The authors have attempted to address many lingering issues; however, the question about the connection between YAP/TAZ and MYC has not been satisfactorily addressed. For example, in Figure 3H, the authors knockout down YAP/TAZ and attempted rescue with control, MYC or mTAZ expression vectors (Fig EV4) on expression of GOT1 (Fig 3H). Looking at just GOT1 does not seem comprehensive without context of other YAP/TAZ targets. Fig EV4 shows that endogenous MYC is diminished with YAP/TAZ, but overexpression of mTAZ did seem to increase endogenous MYC. A longer exposure of the immunoblots in Fig EV4 would help. By contrast, ectopic MYC (Fig EV4) seems diminished by siYAP/TAZ. The effects of mTAZ and siYAP/TAZ on MYC mRNA or protein expression is unclear. However, the apparent rescue of endogenous MYC by mTAZ in the context of siYAP/TAZ suggests a role for MYC as previously documented in several publications. In this regard, the authors' conclusion:"Thus, our results strongly suggest that TAZ/YAP directly induce the expression of GOT1, an enzyme that promotes transamination of glutamate" seems overreaching without the consideration of endogenous MYC levels. The authors did not knockdown endogenous MYC (siMYC) to determine whether MYC is essential for YAP/TAZ regulation of glutamine metabolism. That is, could mTAZ rescue siYAP/TAZ in the presence of siMYC? The authors seem to dismiss the MYC axis without adequate, conclusive experimental evidence. The authors' general findings are still somewhat contradictory to the references, which link YAP/TAZ and MYC, provided in Reviewer 3 issue #3. It is also notable that MYC is bound to many glutamine metabolic genes as found in the ENCODE database that includes MDA-MB-231 cells.

1st Revision - authors' response 19 March 2018

Response to reviewer’s comments:

RESPONSE: We have now included analysis of CTGF expression in our revised Figure 3H, which is a gene that is well defined as a direct target of YAP/TAZ. We show that CTGF expression levels are rescued by ectopic mTAZ, but not ectopic MYC, following YAP/TAZ knockdown. This regulatory pattern is similar to GOT1, supporting our conclusion that GOT1 is directly regulated by YAP/TAZ.

Fig EV4 shows that endogenous MYC is diminished with YAP/TAZ, but overexpression of mTAZ did seem to increase endogenous MYC. A longer exposure of the immunoblots in Fig EV4 would help. By contrast, ectopic MYC (Fig EV4) seems diminished by siYAP/TAZ. The effects of mTAZ and siYAP/TAZ on MYC mRNA or protein expression is unclear. However, the apparent rescue of endogenous MYC by mTAZ in the context of siYAP/TAZ suggests a role for MYC as previously documented in several publications. In this regard, the authors' conclusion: "Thus, our results strongly suggest that TAZ/YAP directly induce the expression of GOT1, an enzyme that promotes transamination of glutamate" seems overreaching without the consideration of endogenous MYC levels.

RESPONSE: As pointed out by the reviewer MYC protein levels do appear to change in response to YAP/TAZ knockdown or following ectopic mTAZ expression. Our observations in Figure 3H also suggest that there are post-transcriptional events regulated by YAP/TAZ that impact MYC levels. One possibility being that MYC levels are affected by YAP/TAZ-mediated regulation of miRNAs that target MYC, such as that proposed in Mori et al. Cell, 2014 (PMID: 24581491). Given this complex relationship it is difficult to rule out the contributions of MYC in our observations, and we have thus revised how we phrase many of the statements throughout our manuscript to account for the relationship with MYC.

The authors did not knockdown endogenous MYC (siMYC) to determine whether MYC is essential for YAP/TAZ regulation of glutamine metabolism. That is, could mTAZ rescue siYAP/TAZ in the presence of siMYC? The authors seem to dismiss the MYC axis without adequate, conclusive experimental evidence. The authors' general findings are still somewhat contradictory to the

references, which link YAP/TAZ and MYC, provided in Reviewer 3 issue #3. It is also notable that MYC is bound to many glutamine metabolic genes as found in the ENCODE database that includes MDA-MB-231 cells.

RESPONSE: As suggested by the reviewer, we have tried knocking down MYC using siRNA in our experiments, but it has been difficult to make strong conclusions based on our observations. One complication being that MYC knockdown reduces TAZ expression (see Reviewer Figure below). Given the potential complexity of the YAP/TAZ-MYC axis, including a posttranscriptional connection, we argue that understanding these mechanisms are beyond the scope of this current manuscript. However, we have revised our manuscript to better discuss the YAP/TAZ-MYC axis and the likely contribution of this axis to the glutamine metabolism. We would like to highlight that regardless of the relationship to MYC our data indicate that YAP/TAZ contribute important roles to glutamine metabolism and that inhibiting glutamine-utilizing transaminases is an effective means for targeting high YAP/TAZ-expressing cancer cells.

Reviewer Figure. MDA-MB-231 cells were transfected with control siRNA or siRNA targeting either MYC or TAZ/YAP and the TAZ gene expression levels were examined by qRT-PCR.

YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND ê USEFUL LINKS FOR COMPLETING THIS FORM PLEASE NOTE THAT THIS CHECKLIST WILL BE PUBLISHED ALONGSIDE YOUR PAPER

Corresponding Author Name: Xaralabos Varelas Journal Submitted to: EMBO Reports Manuscript Number: EMBOR-2016-43577 http://www.antibodypedia.com http://1degreebio.org Reporting Checklist For Life Sciences Articles (Rev. June 2017) http://www.equator-network.org/reporting-guidelines/improving-bioscience-research-reporting-the-arrive-guidelines-for-reporting-animal-research/

This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal’s authorship guidelines in preparing your manuscript. http://grants.nih.gov/grants/olaw/olaw.htm http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm A- Figures http://ClinicalTrials.gov 1. Data http://www.consort-statement.org The data shown in figures should satisfy the following conditions: http://www.consort-statement.org/checklists/view/32-consort/66-title è 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. http://www.equator-network.org/reporting-guidelines/reporting-recommendations-for-tumour-marker-prognostic-studies-remark/ è figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. http://datadryad.org è graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. http://figshare.com è if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified http://www.ncbi.nlm.nih.gov/gap è 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. http://www.ebi.ac.uk/ega

2. Captions http://biomodels.net/ Each figure caption should contain the following information, for each panel where they are relevant: http://biomodels.net/miriam/ è a specification of the experimental system investigated (eg cell line, species name). http://jjj.biochem.sun.ac.za è the assay(s) and method(s) used to carry out the reported observations and measurements http://oba.od.nih.gov/biosecurity/biosecurity_documents.html è an explicit mention of the biological and chemical entity(ies) that are being measured. http://www.selectagents.gov/ è an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

è the exact sample size (n) for each experimental group/condition, given as a number, not a range; è a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.). è a statement of how many times the experiment shown was independently replicated in the laboratory. è definitions of statistical methods and measures: 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.

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.

B- Statistics and general methods Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return)

1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? N was >3 for all experiments. Statistical analyses were performed on each experiment to determine significance of the results.

1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used. N/A

2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre- Samples with clear technical errors, such as uneven seeding, detached during washes, were established? excluded. This criteria was pre-estabolished before samples were processed.

3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. Key experiments were performed and reproduced by different operators. randomization procedure)? If yes, please describe.

For animal studies, include a statement about randomization even if no randomization was used. N/A

4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results N/A (e.g. blinding of the investigator)? If yes please describe.

4.b. For animal studies, include a statement about blinding even if no blinding was done N/A

5. For every figure, are statistical tests justified as appropriate? Yes. See figure legends for the respective statistical tests.

Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Yes

Is there an estimate of variation within each group of data? Yes Is the variance similar between the groups that are being statistically compared? Yes

C- Reagents

6. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog All catalog numbers for the antibodies used are listed in the manuscript methods section. Each number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., antibody was internally validated by immunoblotting lysates from cells treated with siRNA that Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right). knocked down the respective RNA/protein (knockdown was also validated in parrellel using qPCR). 7. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for All cancer cell lines used in this study were purchased from ATCC and stocks were generated for mycoplasma contamination. each cell line. Cells were not passaged for more than one month, and cells were routinely tested for mycoplasma using the Lonza MycoAlert Mycoplasma Detection Kit. * for all hyperlinks, please see the table at the top right of the document

D- Animal Models

8. Report species, strain, gender, age of animals and genetic modification status where applicable. Please detail housing N/A and husbandry conditions and the source of animals.

9. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the N/A committee(s) approving the experiments.

10. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure N/A that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.

E- Human Subjects

11. Identify the committee(s) approving the study protocol. N/A

12. Include a statement confirming that informed consent was obtained from all subjects and that the experiments N/A conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. 13. For publication of patient photos, include a statement confirming that consent to publish was obtained. N/A

14. Report any restrictions on the availability (and/or on the use) of human data or samples. N/A

15. Report the clinical trial registration number (at ClinicalTrials.gov or equivalent), where applicable. N/A

16. For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list at top right) N/A and submit the CONSORT checklist (see link list at top right) with your submission. See author guidelines, under ‘Reporting Guidelines’. Please confirm you have submitted this list. 17. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at N/A top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines.

F- Data Accessibility

18: Provide a “Data Availability” section at the end of the Materials & Methods, listing the accession codes for data N/A generated in this study and deposited in a public database (e.g. RNA-Seq data: Gene Expression Omnibus GSE39462, Proteomics data: PRIDE PXD000208 etc.) Please refer to our author guidelines for ‘Data Deposition’.

Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Functional genomics data e. Proteomics and molecular interactions 19. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the All datasets used are referenced or are included in our Expanded View Tables. journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right). 20. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while N/A respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public access- controlled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right). 21. Computational models that are central and integral to a study should be shared without restrictions and provided in a All computational methods and models have been previously published and are referenced in the machine-readable form. The relevant accession numbers or links should be provided. When possible, standardized mansucript. format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.

G- Dual use research of concern

22. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top There is no concern regarding dual use research for this manuscrupt. right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could.