FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression

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1 Manuscript EMBOR FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression Paraskevi Charitou, Maria Rodriguez-Colman, Johan Gerrits, Miranda van Triest, Marian Groot Koerkamp, Marten Hornsveld, Frank Holstege, Nanda M. Verhoeven-Duif and Boudewijn M.T. Burgering Corresponding author: Boudewijn M.T. Burgering, University Medical Center Utrecht Review timeline: Submission date: 26 May 2014 Editorial Decision: 02 July 2014 Revision received: 26 November 2014 Editorial Decision: 21 December 2014 Revision received: 06 January 2015 Accepted: 08 January 2015 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.) Editor: Esther Schnapp 1st Editorial Decision 02 July 2014 Thank you for the submission of your manuscript to EMBO reports. I apologize for the delay in its decision process, which is also due to recent conference traveling (I was only back in the office yesterday). We have now received the enclosed referee reports on your study, as well as the referee cross-comments on each others' reports. As you will see, all referees acknowledge that the findings are potentially interesting, but also note that the manuscript, as it stands, is too preliminary for publication and that the data need to be significantly strengthened. The referees pinpoint a number of missing important controls, quantifications, statistical analyses and required clarifications. Importantly, referee 1 is asking for several rescue experiments to demonstrate a causal role of FOXO in IDH mediated effects, and vice versa. Both referees 1 and 3 also point out that effects of FOXO deletion on apoptosis, cell cycle and cell differentiation should be investigated. On the other hand, point 7 of referee 1, point 5 of referee 2 and point 4 of referee 3 would not need to be experimentally addressed for consideration of the manuscript for publication here, given the referee cross-comments. From these comments it is clear that publication of the manuscript in our journal cannot be considered at this stage. On the other hand, given the potential interest of your findings, I would like to give you the opportunity to address the concerns and would be willing to consider a revised European Molecular Biology Organization 1

2 manuscript with the understanding that the referee concerns (as mentioned above and in their reports) must be fully addressed and their suggestions taken on board. Should you decide to embark on such a revision, acceptance of the manuscript will depend on a positive outcome of a second round of review. It is EMBO reports policy to allow a single round of revision only and acceptance or rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript. 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: In tumors associated with defects of IDH1/2 enzymes, the common underlying mechanism of tumorigenesis involves the accumulation of an oncometabolite, D-2-HG, which conveys oncogenic signals through driving epigenetic changes and gene regulation. This not only provides strong evidence to support the notion that metabolic dysregulation is the underlying hallmark of cancer, but also represents a fascinating example of the connection among cellular metabolism, chromatin modifications and gene regulation. To date, specific small molecule inhibitors have been developed that efficiently target the enzymatic activity of mutant IDH1/2 and hold promise for the treatment of lower-grade gliomas and AML. Meanwhile, investigating the upstream regulator(s) of IDH1/2, especially their mutants, could potentially be equally beneficial. This study by Paraskevi et al. is directed towards understanding the molecular mechanism by which expression of wild-type and mutant IDH1 genes is regulated. They present evidence indicating that transcription factors of FOXO1 and FOXO3 can induce IDH1 expression, and thereby control cellular α KG production and redox potential. Moreover, they show that FOXO regulate mutant IDH1 expression, and thereby maintain cellular levels of 2-HG, which conveys oncogenic signals through driving epigenetic changes. The current study is potentially interesting, but is seriously limited by the lack of controls in many key experiments, making it difficult to judge the significance. Major Points 1. In this study, the authors have tested the effect of FOXO on regulating IDH1 expression in many cell types. HeLa cells was used to avoid the contribution of p53 dependent transcription, and HT1080 was included which carriers an endogenous mutation of IDH1. Any special reasons for testing the hypothesis in so many other cell lines, such as RPE cells, NIH 3T3 cells overexpressing the insulin receptor (A14 cells), and DL23 cells? If so, please clarify. 2. In Figure 1C and Figure 3B, the western blotting band of FOXO1 is very weak. qrt-pcr analysis is a fairly straightforward experiment in order to verfiy the knockdown efficiency of FOXO. 3. In Figure 1F, the authors demonstrated that the occupancy of FOXO3(A3) on one intron region of IDH1 was increased in DL23 cells upon ER treatment, leading to IDH1 transactivation. As a negative control, stable DL23 cells with FOXO knockdown is needed. 4. In Figure 2A, the authors demonstrated that knocking-down FOXO or IDH1 led to decreased levels of α KG. As negative/positive controls, the intracellular levels of other metabolites is needed, such as isocitrate, glutamate, and TCA metabolites. Moreover, the α KG concentration needs to be normalized by cell protein, considering that doxy treatment or siidh1 will probably inhibit cell growth. In addition, IDH1 put-back control is also needed to demonstrate the decreased α-kg is caused by decreased IDH1 expression by knocking-down FOXO. European Molecular Biology Organization 2

3 5. In Figure 2B, the authors demonstrated that knocking-down FOXO or IDH1 led to reduced [NADPH/NADP+]. As controls, the mrna and protein expression of other NADPH-producing enzymes, such as IDH2, G6PD, MOD1/3, and SHMT1/2 should be tested. In addition, IDH1 putback control is also needed to demonstrate the decreased [NADPH/NADP+] is caused by decreased IDH1 by knocking-down FOXO. 6. In Figure 3C, the authors demonstrated that knocking-down FOXO or IDH1 led to decreased levels of 2-HG, which needs to be normalized by cell protein, considering that doxy treatment or siidh1 will probably inhibit cell growth. In addition, IDH1 put-back control is also needed to demonstrate the decreased 2-HG is caused by decreased IDH1 expression by knocking-down FOXO. 7. In Figure 4A, the authors demonstrated that knocking-down FOXO or IDH1 led to decreased levels of H3K4me3 and H3K9me3. In addition to KDM and TET enzymes, the family of α-kgdependent dioxygenases also include collagen prolyl-4-hydroxylases (CPHs) and HIF prolyl hydroxylases (PHDs). Testing the effects of knocking-down FOXO or IDH1 on Endostatin and HIF1α will be helpful to further support/strengthen the conclusion. 8. In Figure 4B, the dot-blot analysis is NOT convincing, and needs further quantification. In addition to semi-quantification, a more precise method for measuring 5mC and 5hmC contents, such as LC-MS/MS, is needed. Moreover, the 5hmC production in HT1080 should be extremely low, since TET enzymes are largely inactivated in most cultured cancer cell lines. It is hard to believe that knocking-down FOXO or IDH1 would affect 5hmC production catalyzed by TET in HT1080 cells. 9. In Figure 4C and 4D, the authors demonstrated that FOXO deletion led to inhibited cell proliferation and colony formation in HT1080 cells. Is the observed inhibition of cell growth caused by apoptosis or cell cycle arrest? Through altering histone modifications and gene regulation or other mechanisms? Moreover, IDH1 put-back control is needed to demonstrate the inhibited cell growth is caused by decreased IDH1 expression by knocking-down FOXO. Referee #2: The manuscript by Charitou et al., aims at demonstrating that transcription factors FOXOs regulate the isocitrate dehydrogenases (IDH)1 expression both in normal and in cancer cells. The finding that FOXOs control IDH1 expression is very interesting and in line with recently increased reports on metabolic functions of FOXOs. However there are several points that need to be addressed. 1) The data is relatively heterogeneous and distinct parts are derived from several distinct lines (Hela, RPE, DL23, HT10800). The authors should at least confirm some of the major findings in the same line. It seems that RPEshFOXO is actually shfoxo1. The authors should make that clear. How many lines were derived? Is the data from one line or multiple lines? They should also quantify the expression of FOXO1 and IDH1 in Fig. 1C. The labeling of 1C (IDH1 sirna) is misleading as this is in RPEshFOXO cells. Fig. 1D, it is not clear how sirna were introduced in cells (transfection?) and what is the efficiency. The specificity of sirna or shrna against each FOXO in these experiments needs to be addressed. It is also important to include more than one sirna for each target. 2) Fig. 2A: the levels of expression of FOXO (FOXO1?) or IDH1 should be shown. What is the efficiency of sirna IDH1? 3) Fig. 3B. information regarding levels of knockdown of FOXO1 is missing. It is hard to assess the levels of FOXO1 on this Western blot. Overall what is referred to as FOXO depleted cells shows decreased expression (50%?). Knock down FOXO followed by level of knockdown (50-60% for instance would be more appropriate. 4) It is hard to assess how meaningful the results of Fig. 4A are. Can the authors provide standard European Molecular Biology Organization 3

4 deviation for the results of the three experiments? 5) What is the status of endogenous FOXO1 and/or FOXO3 subcellular localization/activity in HT1080 cells. Does endogenous FOXO regulate IDH1 expression? The data presented does not support necessarily "On the other hand, in transformed cells carrying IDH1 mutation, FOXOs are required for the maintenance of mutant IDH1 levels and thereby for the levels of the generated". These engineered cells are not representing the physiological setting of cancer cells in vivo. Although the authors show that FOXO regulate the expression of mutant IDH1, these data do not support FOXO induction of mutant IDH1 expression in cancer cells in vivo. In general the use of terminology of physiological setting in the context of experimental system used in this paper does not seem appropriate. Referee #3: Charitou et al. use stable doxycycline-inducible shrnas targeting FOXO transcription factors to analyze gene expression changes upon knockdown of FOXOs. The authors find that IDH1 expression is reduced upon FOXO knockdown and that this corresponds to a reduction in α- ketoglutarate and the NADPH/NADP+ ratio. In addition, they demonstrate that FOXOs regulate the expression of mutant IDH1 in transformed cells, in turn affecting the production of 2-HG. In these cells, knockdown of FOXO transcription factors also leads to reduced cell growth and colony formation in soft agar. The authors propose a dual role for FOXO transcription factors depending on the cellular context. In untransformed cells, FOXOs regulate the expression of wild-type IDH1 to maintain α-ketoglutarate and NADPH levels, contributing to the production of glutathione to buffer against ROS. However, in the context of transformed cells, FOXOs are also capable of regulating the expression of mutant IDH1 and thereby the production of 2-HG to support malignancy. The idea that FOXO transcription factors can regulate the expression of IDH1 is an interesting finding and one that could be potentially useful for developing cancer therapies. However, the experiments lack a control for doxycycline and some of the data suggests that off-target effects, rather than changes in IDH1 expression, may be contributing to some of the phenotypes observed. Overall, although the underlying concept may be interesting to the cancer metabolism community, the data seem preliminary and the manuscript does not do enough to put the findings into context of current models for 2HG function. Major Comments 1. The authors use a doxycycline-inducible system throughout the paper but have no control to distinguish the effects of doxycycline from changes in FOXO gene expression. Doxycycline can have major metabolic effects in cell culture (see Ahler et al, PLOSOne 2013, PMID ). All experiments using this system should include a nonsense hairpin with doxycycline to control for these effects. Additionally, a second FOXO shrna should be used in at least a subset of the critical experiments. 2. In Figure 1A, the level of FOXO knockdown achieved with this doxcycline-inducible system should be provided. 3. The experiment in Figure 2B is irrelevant. Withdrawing glucose will elicit a very large number of changes that will complicate the interpretation. The reader gets the impression that IDH silencing has essentially no effect on the NADPH/NADP+ ratio when glucose is present. Is that the case? 4. In Figure 4, the differences between the doxycycline-inducible FOXO knockdown and IDH1 knockdown suggest that off-target effects may be playing a role in some of these observations. Specifically in Figure 4A, IDH1 sirna yields a better knockdown of IDH1 protein, but does not result in greater reduction of histone methylation compared to the FOXO knockdown. The same is true in Figure 4B, where the addition of doxycycline results in an increase in 5- hydroxymethylcytosine, whereas the IDH1 knockdown does not show this effect. This suggests that the FOXO system may have some off-target effects contributing to some of the phenotypes observed. 5. FOXO transcription factors have many targets. To verify that the growth reduction and soft agar colony reduction in Figure 4C and 4D are due to IDH1 loss, the authors should include the IDH1 sirna knockdown as a positive control, as they have done for previous experiments. 6. Rather than simply studying histone methylation, the authors should provide some specific details about how gene expression is altered. The role of 2HG in regulating gene expression is now well European Molecular Biology Organization 4

5 established. The prevailing model is that the oncometabolite suppresses the induction of differentiation programs to keep the cell is a relatively de-differentiated state. Are these programs also altered in the authors' system? Do the cells activate a differentiation program when FOXOs or IDH1 are silenced? 7. Crucially, in the HT1080 model, the authors are silencing BOTH wild-type and mutant IDH1. How can they be sure that the effects on growth are related to mutant IDH1 silencing, or depletion of 2-HG? They should attempt to reverse these findings by adding back 2-HG. Minor Comments 1. FOXOs are well-known regulators of metabolic enzyme expression, particularly antioxidant enzymes. Did these canonical targets score in the analysis used here? 2. The authors claim that 2-HG is required for survival (Abstract). No data on survival or cell death are presented in the paper. As discussed above, 2-HG is thought to interfere with cellular differentiation, not to provide a survival advantage. 3. The authors use the terms tumor suppressor and oncogene too loosely. In several places, they claim that FOXOs are "bona fide tumor suppressors." This is based on the observation that knocking out multiple FOXOs enhances tumor formation in mice. Being a "bona fide" tumor suppressor implies that FOXOs are mutated, deleted, or otherwise silenced in human cancer. In the discussion and in Figure 5, the authors use the word oncogene in reference to FOXOs. This description is not really appropriate given that FOXOs are not oncogenes; they regulate the expression of mutant IDH1 protein in a transformed cellular context. 4. A better description of DL23 cells and the use of the system in ChIP experiments should be provided. 5. In the discussion, the authors reference a Figure 6, but there are only five figures in this paper. Cross-comments Referee 1: Reviewer 2 point 5: Agree. This comment regarding the potential role of endogenous FOXO in regulating mutant IDH1 expression is valuable, and does need to be addressed. Reviewer 3 point 4: Although IDH1 sirna yields a better knockdown of IDH1 protein than the doxycycline-inducible FOXO knockdown, it may not change the intracellular levels of α-kg and 2- HG (as shown in Fig. 3C). This may explain the observed similar effect of FOXO knockdown and IDH1 knockdown on changing epigenetic markers. The authors should discuss this possibility in the text. Reviewer 3 point 6: Agree. As pointed out by myself (Review 1#, point 9), there is clearly a gap between histone methylation alteration and cell proliferation change. The authors need to address this point by performing additional experiments to test cell cycle, apoptosis, ect, and more importantly, the association between epigenetic changes and cell growth in the authors' system should be futher discovered. Referee 2: I don't find the scope of this paper calls for point 7 of the reviewer 1 or point 6 of the reviewer 3. The explanation for point 4 of reviewer 3 might be that the knockdown of FOXO has additional effects as compared to knockdown of IDH1 on histone methylation (not just through IDH1). The authors can perhaps discuss this point and mechanisms that might explain differences between the effects of FOXO and IDH1 knock down. Overall I agree with other reviewers that the data provided does support the "labeling" of FOXO as an oncogene. Referee 3: Reviewer 1, Point 7: This is a logical question, but it would provide tangential data. I would not demand that they provide additional data. The role of mutant IDH1 in regulating epigenetics is on much more solid ground than its effects on prolyl hydroxylation, so I think the authors have already chosen the most informative targets. European Molecular Biology Organization 5

6 Reviewer 2, Point 5: This is a very convoluted question. The reviewer is correct that the situation does not represent the physiological setting of a tumor growing in vivo, but the same criticism could be applied to 90% of the papers in cancer metabolism, including the role of IDH1 in epigenetic reprogramming. Furthermore, the authors did demonstrate that FOXO silencing reduced IDH1 expression and 2HG formation, so I think the role of FOXOs in regulating mutant IDH1 expression is fairly clear in this system. 1st Revision - authors' response 26 November 2014 Point-by-point response Referee #1: Major Points 1. In this study, the authors have tested the effect of FOXO on regulating IDH1 expression in many cell types. HeLa cells was used to avoid the contribution of p53 dependent transcription, and HT1080 was included which carriers an endogenous mutation of IDH1. Any special reasons for testing the hypothesis in so many other cell lines, such as RPE cells, NIH 3T3 cells overexpressing the insulin receptor (A14 cells), and DL23 cells? If so, please clarify. The choice of cell lines is because (-) We wished to establish whether regulation of IDH1 by FOXOs was cancer cell line specific or also occurs in normal untransformed RPE cells. (-) We wished to establish whether regulation of IDH1 is also observed in a setting of inhibition of FOXO by normal PI3K/PKB(AKT) signaling rather than the (still artificial) inhibition of endogenous FOXO by sirna/shrna. Therefore the use of A14 cells, which is an insulin sensitive NIH3T3 cell line we have used/described in the past (ref: Burgering et al. EMBO J EMBO J May;10(5): ) now provided in the text). (-) As we have all Chip-seq data obtained in the DL23 cells (expressing 4-OHT inducible FOXO3(A3)) we used these cells to directly translate recruitment of FOXO to chromatinized DNA to regulation of IDH1 expression Finally, on a more general note, similar to c-myc we and others have noticed that FOXO-dependent gene regulation appears context (cell type)-dependent (discussed in Eijkelenboom et al. Cell reports 2013). Thus testing more than one cell line provides information as to whether IDH1 regulation is also cell context dependent (or not). We have rewritten parts of the text as to clarify better for the general reader the rationale for using these cell lines 2. In Figure 1C and Figure 3B, the western blotting band of FOXO1 is very weak. qrt-pcr analysis is a fairly straightforward experiment in order to verfiy the knockdown efficiency of FOXO. The requested qrt-pcr is added also for IDH1 and figure is now extended to also include a luciferase shrna control. Western blot has been replaced (new figure 1B and 1C as well as 3A and 3B) 3. In Figure 1F, the authors demonstrated that the occupancy of FOXO3(A3) on one intron region of IDH1 was increased in DL23 cells upon ER treatment, leading to IDH1 transactivation. As a negative control, stable DL23 cells with FOXO knockdown is needed. In two of our previous studies studying Chip-seq of FOXO3a (Eijkelenboom et al. Cell Rep Dec 26;5(6): and Mol Syst Biol. 2013;9:638.) we have used the following controls besides IgG: (-) DLD1 cells with (+) or without (+) 4OHT treatment. DLD1 is the parental cell line for DL23 without the 4OHT inducible FOXO3a(A3) construct so in essence similar to the control asked for (DL23 with FOXO3 knockdown) by the reviewer. The track for DLD1 +4OHT is provided in figure European Molecular Biology Organization 6

7 1E and there is no enrichment above basal using the antibody to precipitate FOXO3(A3). (-) We have performed Chip-seq of FOXO3(A3) lacking the DNA binding domain which also results in lack of enrichment (Eijkelenboom et al. Mol Syst Biol. 2013;9:638). We believe that our previous studies to which we refer, sufficiently demonstrate the specificity of our approach and combined with the Chip-qRT-PCR data show that FOXO3a upon 4OHT treatment is recruited to the IDH1 gene and that this correlates with regulation of IDH1 expression. 4. In Figure 2A, the authors demonstrated that knocking-down FOXO or IDH1 led to decreased levels of α KG. As negative/positive controls, the intracellular levels of other metabolites is needed, such as isocitrate, glutamate, and TCA metabolites. We thank the reviewer for this suggestion, as this appeared an informative experiment. We have measured the following additional metabolites: succinate, fumarate, glutamine glutamate. These data are now represented in Figure 2 B. Glutamine is reduced which is in agreement with our previous study showing FOXO regulation of Glutamine synthase (van der Vos et al. Nat Cell Biol Aug;14(8):829-37). However this does unlikely contribute to reduced a-kg as glutamate levels remain unchanged. The change in citrate and isocitrate we cannot explain directly but we noted that reductive carboxylation involves the flow from glutamine to citrate so the coupling of these metabolites following FOXO knockdown we may take to suggest a possible role for FOXOs in regulating reductive carboxylation a possibility we are currently studying further. Moreover, the α KG concentration needs to be normalized by cell protein, considering that doxy treatment or siidh1 will probably inhibit cell growth. In all measurements metabolite concentrations were normalized to cell number, which we believe equally corrects for possible changes in cell proliferation, which is a known effect of FOXOs. This is now indicated in all figure concerned. In addition, IDH1 put-back control is also needed to demonstrate the decreased a-kg is caused by decreased IDH1 expression by knocking-down FOXO. We have performed the add-back (new Figure 2A) and a-kg level is rescued showing that FOXOs regulate a-kg through IDH1. 5. In Figure 2B, the authors demonstrated that knocking-down FOXO or IDH1 led to reduced [NADPH/NADP+]. As controls, the mrna and protein expression of other NADPH-producing enzymes, such as IDH2, G6PD, MOD1/3, and SHMT1/2 should be tested. We tested IDH2, G6PD, MOD1/3. G6PD levels remain constant whereas IDH2, MOD1 and 2 mrnas increase following FOXO knockdown (new Figure 2D). MOD3 we could not detect in these cells. Thus these enzymes are unlikely involved in the observed reduction of NADPH, but rather are activated as part of a compensatory mechanism. In addition, IDH1 put-back control is also needed to demonstrate the decreased [NADPH/NADP+] is caused by decreased IDH1 by knocking-down FOXO. In agreement with the above following IDH1 add back not only a-kg but also NADPH levels are restored. Combined this shows that the decrease in NADPH is due to decreased IDH1 following FOXO knockdown (new Figure 2C) 6. In Figure 3C, the authors demonstrated that knocking-down FOXO or IDH1 led to decreased levels of 2-HG, which needs to be normalized by cell protein, considering that doxy treatment or siidh1 will probably inhibit cell growth. Indeed siidh1 and shfoxo inhibits cell growth (new figure 4D and 4E) and as for the other experiments we corrected for cell number rather than protein. In addition, IDH1 put-back control is also needed to demonstrate the decreased 2-HG is caused by decreased IDH1 expression by knocking-down FOXO. European Molecular Biology Organization 7

8 We have tried extensively to generate add-back cell lines in HT1080 (transient ectopic expression is not really an option), however this requires stable expression of both wt IDH1 as well mutant IDH1. Although we obtained clones these clones never expressed the ectopic (tagged) IDH1. We have no explanation as to why this does not appear to work for HT1080 cancer cells whereas add-back expression of wt IDH1 only, in untransformed RPE cells does work. Apparently there is a selection against (over)expression of both mutant and wt IDH1 in HT1080 cells. However we consider it unlikely (mechanistically) that FOXOs would regulate only the wt IDH1 allele and not the mutant IDH1 allele. 7. In Figure 4A, the authors demonstrated that knocking-down FOXO or IDH1 led to decreased levels of H3K4me3 and H3K9me3. In addition to KDM and TET enzymes, the family of a-kgdependent dioxygenases also include collagen prolyl-4-hydroxylases (CPHs) and HIF prolyl hydroxylases (PHDs). Testing the effects of knocking-down FOXO or IDH1 on Endostatin and HIF1a will be helpful to further support/strengthen the conclusion. Based on cross-referee comments and suggestion of the editor we have not further investigated this matter in the context of the current study. 8. In Figure 4B, the dot-blot analysis is NOT convincing, and needs further quantification. In addition to semi-quantification, a more precise method for measuring 5mC and 5hmC contents, such as LC-MS/MS, is needed. Moreover, the 5hmC production in HT1080 should be extremely low, since TET enzymes are largely inactivated in most cultured cancer cell lines. It is hard to believe that knocking-down FOXO or IDH1 would affect 5hmC production catalyzed by TET in HT1080 cells. Indeed TET levels are low and consequently signals and consequent changes are low. To in part circumvent this we ectopically expressed TET1 and observed again an induction of 5hmC following FOXO knockdown, which importantly is not observed when cell are exogenously treated with cell permeable 2-HG (Obtained from Dr Ryan Looper; Supplementary Figure 4A). We appreciate the suggestion to use mass-spectrometry to quantify signals but we consider this outside the scope of our paper, firstly because our report is not intended to show that a-kg is a co-factor for TET enzymes, this has been shown by numerous other studies and second this approach is far from trivial and would require a considerable investment in time (and money) that would greatly exceed the time allowed for revision. 9. In Figure 4C and 4D, the authors demonstrated that FOXO deletion led to inhibited cell proliferation and colony formation in HT1080 cells. Is the observed inhibition of cell growth caused by apoptosis or cell cycle arrest? Cell number is the combined consequence of proliferation and cell death(apoptosis). We now show that loss of FOXO induces apoptosis, whereas loss of IDH1 has no significant effect on apoptosis. Because addition of 2-HG rescues in part cell number and IDH1 loss reduces cell number we conclude that FOXOs through IDH1 regulate proliferation. Whether or not this is because FOXOs and 2-HG maintain a de-differentiated state, as suggested by literature, we discuss but have not further explored. Mostly because we do not know what the differentiation program would be for HT1080 cells. (new Figure 4D, 4E, 4F and Supplementary Figure 4B). Through altering histone modifications and gene regulation or other mechanisms? FOXOs control many mechanisms for cell proliferation, apoptosis and survival. We believe that pinpointing what mechanism(s) causally underlies this goes beyond the scope of our study. We provide correlative data on epigenetics and, as far as we know, this reflects the current status of the field. Again as far as known to us, also for the control of cell growth by IDH1/2-HG the correlative data are clear yet a causal mechanism remains still to be defined. Moreover, IDH1 put-back control is needed to demonstrate the inhibited cell growth is caused by decreased IDH1 expression by knocking-down FOXO. See also answer above, we were unable to obtain add-back cell lines for HT1080 but instead have European Molecular Biology Organization 8

9 functionally restored IDH1 expression by exogenous addition of cell permeable 2-HG. Referee #2: However there are several points that need to be addressed. 1) The data is relatively heterogeneous and distinct parts are derived from several distinct lines (Hela, RPE, DL23, HT10800). The authors should at least confirm some of the major findings in the same line. See our answer to reviewer #1 concerning the rationale for using different cell lines. Furthermore with the additional experiments we believe that we confirmed most of our findings within RPE cells, except of course for the regulation of mutant IDH1 It seems that RPEshFOXO is actually shfoxo1. The authors should make that clear We apologize for not being clear here, but the inducible shrna targets both FOXO3 and FOXO1. Sometimes we additionally target FOXO4 by an independent sirna but the cell lines we have used here do not express FOXO4 at sufficient level. This is stated in the text. How many lines were derived? Is the data from one line or multiple lines? The cell lines are polyclonal. Some but not all experiments have been done on at least two independent (polyclonal) cell lines. They should also quantify the expression of FOXO1 and IDH1 in Fig. 1C. The labeling of 1C (IDH1 sirna) is misleading as this is in RPEshFOXO cells. Fig. 1D, it is not clear how sirna were introduced in cells (transfection?) and what is the efficiency. The specificity of sirna or shrna against each FOXO in these experiments needs to be addressed. It is also important to include more than one sirna for each target. The issues raised are clarified in the text. Labeling was indeed incorrect but also upon request of reviewer #1 we have extended and improved this figure (now figure 1B and 1C). We have indicated in the materials and method section how sirna was introduced (transfection) and efficiency (normally >90%). The sirnas and the shrna have been extensively described in previous literature by others and us including controls for off-target (FOXO add-back) etc. (e.g. Potente et al. J Clin Invest Sep;115(9): ; de Keizer et al. Cancer Res Nov 1;70(21): Hribal et al J Cell Biol Aug 18;162(4): ). In addition we would like to note that when we inhibit endogenous FOXO by insulin signaling through PI3K/AKT we also observe IDH1 regulation. Also silencing FOXOs individually (Figure 1D) in essence is an experiment with multiple sirnas. 2) Fig. 2A: the levels of expression of FOXO (FOXO1?) or IDH1 should be shown. What is the efficiency of sirna IDH1? This experiment has been done in RPE cells and we show a typical result of knockdown in these cells in new Figure 1C. Inter-experimental variation left aside knockdown almost always ranges between 60-90%. 3) Fig. 3B. information regarding levels of knockdown of FOXO1 is missing. It is hard to assess the levels of FOXO1 on this Western blot. We have repeated this experiment to also include additional requested controls and provide (we think) better data (new Figure 3A and B) that allow assessment of FOXO1 level. Overall what is referred to as FOXO depleted cells shows decreased expression (50%?). Knockdown FOXO followed by level of knockdown (50-60% for instance would be more appropriate. European Molecular Biology Organization 9

10 We have adjusted the text where appropriate to specify the level of knockdown. However, including this in all cases where we quote FOXO knockdown would render a rather unreadable whole. 4) It is hard to assess how meaningful the results of Fig. 4A are. Can the authors provide standard deviation for the results of the three experiments? This experiment was performed 3 times with essentially similar results. Standard deviation is indicated in the figure legend. 5) What is the status of endogenous FOXO1 and/or FOXO3 subcellular localization/activity in HT1080 cells. Does endogenous FOXO regulate IDH1 expression? The data presented does not support necessarily "On the other hand, in transformed cells carrying IDH1 mutation, FOXOs are required for the maintenance of mutant IDH1 levels and thereby for the levels of the generated". These engineered cells are not representing the physiological setting of cancer cells in vivo. Although the authors show that FOXO regulate the expression of mutant IDH1, these data do not support FOXO induction of mutant IDH1 expression in cancer cells in vivo. In general the use of terminology of physiological setting in the context of experimental system used in this paper does not seem appropriate. The status of endogenous FOXO1 and FOXO3 subcellular localization is now shown in Supplementary Figure 3A. As knockdown of endogenous FOXOs (1 and 3 simultaneously) results in loss of IDH1 expression, we therefore conclude that endogenous FOXOs regulate IDH1 expression. With respect to the rest of the comments we refer to the cross-referee comments and the suggestion of the editor to not further address this. Referee #3: Major Comments 1. The authors use a doxycycline-inducible system throughout the paper but have no control to distinguish the effects of doxycycline from changes in FOXO gene expression. Doxycycline can have major metabolic effects in cell culture (see Ahler et al, PLOSOne 2013, PMID ). All experiments using this system should include a nonsense hairpin with doxycycline to control for these effects. The reviewer is correct that doxycycline may have side effects. We have therefore repeated all relevant experiments and included for these shluciferase expressing cells (-/+ doxycycline) as control (see new Fig 2A, 2B, 3A,3B, 4E, 4F) At least with respect to the parameters measured in these experiments we do not observe any significant contribution of doxycycline addition to the measured effects. Additionally, a second FOXO shrna should be used in at least a subset of the critical experiments. See answer to reviewer #2 2. In Figure 1A, the level of FOXO knockdown achieved with this doxcycline-inducible system should be provided. Is now provided (see Supplementary Figure 1A) 3. The experiment in Figure 2B is irrelevant. Withdrawing glucose will elicit a very large number of changes that will complicate the interpretation. The reader gets the impression that IDH silencing has essentially no effect on the NADPH/NADP+ ratio when glucose is present. Is that the case? The reviewer is correct that glucose withdrawal may complicate interpretation. Therefore, we have repeated this particular experiment (without glucose, but with glutamine and pyruvate) and compared this to with glucose (Supplementary Figure 2C ). As expected the change in NADPH/NADP+ becomes less (due to the contribution of the PPP), but remains clear and significant. Thus also in the presence of glucose IDH1 reduction still affects the NADPH/NADP+ ratio. In retrospect we learned that apparently the contribution of the PPP to the maintenance of NADPH levels is less than we anticipated. European Molecular Biology Organization 10

11 4. In Figure 4, the differences between the doxycycline-inducible FOXO knockdown and IDH1 knockdown suggest that off-target effects may be playing a role in some of these observations. Specifically in Figure 4A, IDH1 sirna yields a better knockdown of IDH1 protein, but does not result in greater reduction of histone methylation compared to the FOXO knockdown. The same is true in Figure 4B, where the addition of doxycycline results in an increase in 5- hydroxymethylcytosine, whereas the IDH1 knockdown does not show this effect. This suggests that the FOXO system may have some off-target effects contributing to some of the phenotypes observed. FOXOs are transcription factors that regulate a multitude of genes and cellular processes and thus rather than to suggest off-target effects we would suggest that FOXO knockdown affects other processes that ultimately may impinge on the same read-out (histone modifications) as IDH1. With these experiments it is not our contention to suggest that IDH1 should be the only mechanism whereby FOXOs regulate epigenetic programs. We believe this notion is also indicated in crossreferee comments by reviewer #1 Although IDH1 sirna yields a better knockdown of IDH1 protein than the doxycycline-inducible FOXO knockdown, it may not change the intracellular levels of α-kg and 2-HG (as shown in Fig. 3C). This may explain the observed similar effect of FOXO knockdown and IDH1 knockdown on changing epigenetic markers. 5. FOXO transcription factors have many targets. To verify that the growth reduction and soft agar colony reduction in Figure 4C and 4D are due to IDH1 loss, the authors should include the IDH1 sirna knockdown as a positive control, as they have done for previous experiments. This has now been done for growth reduction (see new figure 4 D) 6. Rather than simply studying histone methylation, the authors should provide some specific details about how gene expression is altered. The role of 2HG in regulating gene expression is now well established. The prevailing model is that the oncometabolite suppresses the induction of differentiation programs to keep the cell is a relatively de-differentiated state. Are these programs also altered in the authors' system? Do the cells activate a differentiation program when FOXOs or IDH1 are silenced? The additional experiments now done in answer to reviewer #1 lead us to conclude that FOXO regulation of IDH1 indeed may impact on proliferation (New Figure 4 D,E and F) 7. Crucially, in the HT1080 model, the authors are silencing BOTH wild-type and mutant IDH1. How can they be sure that the effects on growth are related to mutant IDH1 silencing, or depletion of 2-HG? They should attempt to reverse these findings by adding back 2-HG. Indeed the reviewer is right, IDH1 functions as a tetramer and it has been shown that for mutant IDH1 to function it requires wt IDH1. Thus formally a reduction in 2-HG can also be achieved by loss of wt IDH1 only. However, we cannot envision a mechanism whereby a transcription factor (here FOXO) can discriminate between the wt and the mutant allele with respect to promoter binding, RNA polii recruitment etc. Irrespective, to address this issue we have obtained cell permeable 2-HG from Dr Ryan Looper. We observe that 2-HG can alleviate the proliferative defect in part, and in agreement IDH1 knockdown (targeting both wt and mutant allele) by itself also results in impaired growth. Thus we conclude that the FOXO effect on growth at least in part is related to its regulation of IDH1 and the production of 2-HG. We wish to note that we do want to provide the impression that IDH1 regulation by FOXOs is the only mechanism by which FOXO mediates effects on growth, as several other mediators in this respect have been described (p27, p21, TORC2 etc.). Minor Comments 1. FOXOs are well-known regulators of metabolic enzyme expression, particularly antioxidant enzymes. Did these canonical targets score in the analysis used here? Yes several of the known FOXO target genes (MnSOD,sestrin3) involved in redox did show changes European Molecular Biology Organization 11

12 in mrna expression following FOXO knockdown, but these were not called significant in this microarray experiment, we did not elaborate on these as these target genes have been published by us and others. 2. The authors claim that 2-HG is required for survival (Abstract). No data on survival or cell death are presented in the paper. As discussed above, 2-HG is thought to interfere with cellular differentiation, not to provide a survival advantage. The reviewer is correct and we have rephrased this in our abstract. In answer to reviewer #1 we have further analysed cell growth/apoptosis and this analysis does now show that in mutant IDH1 cells FOXO regulation of IDH1 and 2-HG contributes to proliferation and this is likely a result of interfering with differentiation. 3. The authors use the terms tumor suppressor and oncogene too loosely. In several places, they claim that FOXOs are "bona fide tumor suppressors." This is based on the observation that knocking out multiple FOXOs enhances tumor formation in mice. Being a "bona fide" tumor suppressor implies that FOXOs are mutated, deleted, or otherwise silenced in human cancer. In the discussion and in Figure 5, the authors use the word oncogene in reference to FOXOs. This description is not really appropriate given that FOXOs are not oncogenes; they regulate the expression of mutant IDH1 protein in a transformed cellular context. We agree with the reviewer and have rephrased the text to be more precise in this matter. We adopted the term bona fide tumor suppressors indeed from Tothova et al. and and we agree with the reviewer that FOXOs are not tumor suppressors senso stricto. However, PI3K activation, which is frequent in tumors can result in functional inhibition of FOXOs and this can be considered to be in compliance with what the reviewer refers to as otherwise silenced in human cancer. Similarly, we agree that FOXOs are not classical oncogenes but indeed regulate the expression of mutant IDH1 protein in a transformed cellular context. Irrespective the functional consequence is that FOXOs are required for tumor cells to survive and thus sustain oncogenic potential. We have adjusted our text to not use these terms too loosely. 4. A better description of DL23 cells and the use of the system in ChIP experiments should be provided. Done 5. In the discussion, the authors reference a Figure 6, but there are only five figures in this paper. Corrected, should have read figure 5 Cross-comments Referee 1: Reviewer 2 point 5: Agree. This comment regarding the potential role of endogenous FOXO in regulating mutant IDH1 expression is valuable, and does need to be addressed. Reviewer 3 point 4: Although IDH1 sirna yields a better knockdown of IDH1 protein than the doxycycline-inducible FOXO knockdown, it may not change the intracellular levels of α-kg and 2- HG (as shown in Fig. 3C). This may explain the observed similar effect of FOXO knockdown and IDH1 knockdown on changing epigenetic markers. The authors should discuss this possibility in the text. Reviewer 3 point 6: Agree. As pointed out by myself (Review 1#, point 9), there is clearly a gap between histone methylation alteration and cell proliferation change. The authors need to address this point by performing additional experiments to test cell cycle, apoptosis, ect, and more importantly, the association between epigenetic changes and cell growth in the authors' system should be futher discovered. Referee 2: European Molecular Biology Organization 12

13 I don't find the scope of this paper calls for point 7 of the reviewer 1 or point 6 of the reviewer 3. (1 and 3 agree on this The explanation for point 4 of reviewer 3 might be that the knockdown of FOXO has additional effects as compared to knockdown of IDH1 on histone methylation (not just through IDH1). The authors can perhaps discuss this point and mechanisms that might explain differences between the effects of FOXO and IDH1 knock down. Overall I agree with other reviewers that the data provided does support the "labeling" of FOXO as an oncogene. Referee 3: Reviewer 1, Point 7: This is a logical question, but it would provide tangential data. I would not demand that they provide additional data. The role of mutant IDH1 in regulating epigenetics is on much more solid ground than its effects on prolyl hydroxylation, so I think the authors have already chosen the most informative targets. Reviewer 2, Point 5: This is a very convoluted question. The reviewer is correct that the situation does not represent the physiological setting of a tumor growing in vivo, but the same criticism could be applied to 90% of the papers in cancer metabolism, including the role of IDH1 in epigenetic reprogramming. Furthermore, the authors did demonstrate that FOXO silencing reduced IDH1 expression and 2HG formation, so I think the role of FOXOs in regulating mutant IDH1 expression is fairly clear in this system. 2nd Editorial Decision 21 December 2014 Thank you for the submission of your revised manuscript to our journal. We have now received the enclosed reports from the referees. As you will see, while referee 1 is rather critical and points out that the data show that Foxo does not control cell growth and apoptosis through IDH1, the other 2 referees agree, also after having seen each other's comments, that the manuscript reports interesting findings and that it can be accepted for publication. We can therefore in principle accept your paper. However, given the critical comments, I would like to ask you to please go carefully again through the manuscript text and modify all sentences that may give the wrong impression that Foxo regulates cell growth and apoptosis through IDH1. Please also address the minor concern of referee 1. The manuscript text is a little bit on the long side. You can either combine the results and discussion sections, or just shorten the text, for example the discussion. I would also like to suggest to modify the before-last sentence in the abstract to: "In cancer cells carrying mutant IDH1, FOXOs likewise regulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation." Please let me know whether you agree with this change. Regarding statistics, not all figure legends specify "n" (for the number of independent experiments, but not duplicates/replicates), the bars (e.g. mean) and error bars (e.g. SEM, SD). Please add this information to all relevant figure panels. Please also note that no error bars can be shown when n=2 (currently specified for figure panels 2B, 3C, 4F). When n=2 please show the actual data points of the 2 experiments in the graph along with the mean. Please also add a scale bar to all microscopy images, e.g. SF3A. I am out of the office now until the 5th of January, so you have time to send us the revised, final manuscript until early January. European Molecular Biology Organization 13

14 REFEREE REPORTS: Referee #1: The new set of experiments improved the manuscript at some points, for instance more convincingly demonstrating that FOXOs selectively regulate IDH1 expression and thereby control NADPH homeostasis and redox potential in the cell. But, Figure 4 still remains major concerns. The authors demonstrated that FOXO knockdown (HT1080shFOXO+doxy) almost entirely suppressed cell growth (Fig. 4D) and increased apoptosis (Fig. 4F). In contrast, the effects of IDH1 knockdown (HT1080shFOXO+siIDH1) on cell growth and apoptosis are minor. It is hard to believe that the observed effect of FOXO knockdown on changing cell number (cell proliferation and apoptosis) is mediated by IDH1 down-regulation (due to FOXO knockdown). Moreover, the authors provided data showing that HT1080shFOXO+doxy (=FOXO knockdown) and HT1080shFOXO+siIDH1(=IDH1 knockdown) have almost the same effect on reducing the oncometabolite 2-HG (Fig. S3B). Why did these two types of cells with almost the same levels of 2- HG accumulation, behave so differently in cell growth and apoptosis? As the authors have pointed out, "FOXOs control many mechanisms for cell proliferation, apoptosis and survival". Based on the functional assay in Fig. 4D-F, it seems that the effect of FOXO knockdown on suppressing cell growth and increasing cell apoptosis is NOT associated with IDH1 down-regulation and 2-HG change. Minor concern: In Fig. 4B, the presence of ectopically expressed TET1 should be shown. Is TET1 overexpressed at an equal level among three samples, NT vs. +doxy vs. IDH1 sirna? Referee #2: The manuscript by Charitou et al., is highly improved. The authors have largely responded to reviewers' concerns. Their data suggest that although loss of FOXO like loss of IDH1 impairs cell growth, the impact of loss of FOXO on cell growth is superior to that of loss of IDH1. This might be due in part to lesser efficacy of sirna targeting IDH1 as compared to FOXO inhibition and/or more likely to a broader impact of loss of FOXO on both cell proliferation and apoptosis as they show. The authors did not investigate in more detail the proliferation status of cells in response to loss of FOXO versus loss of IDH1. However their results as presented are overall supportive of FOXO regulation of IDH1 expression contributing to cell growth. This is an important finding with significant implications. Referee #3: I am satisfied with the authors' revision of the manuscript. Cross-comments from Referee 3: The Referee's points are fair, but I think the authors did enough to support the argument that FOXOmediated regulation of IDH1 contributes part of the overall effects of FOXOs on cell survival/growth. They admit that FOXO transcription factors will undoubtedly elicit other effects besides IDH1 expression, but I feel that exploring all of these effects are beyond the scope of an EMBOR paper. As it stands, the paper reports a focused but interesting aspect of FOXO and IDH1 biology. I think it is worth publishing the paper. European Molecular Biology Organization 14