Targeting prominin2 transcription to overcome ferroptosis resistance in cancer

Abstract Understanding how cancer cells resist ferroptosis is a significant problem that impacts ongoing efforts to stimulate ferroptosis as a therapeutic strategy. We reported that prominin2 is induced by ferroptotic stimuli and functions to resist ferroptotic death. Although this finding has significant implications for therapy, specific prominin2 inhibitors are not available. We rationalized that the mechanism by which prominin2 expression is induced by ferroptotic stress could be targeted, expanding the range of options to overcome ferroptosis resistance. Here, we show that that 4‐hydroxynonenal (4HNE), a specific lipid metabolite formed from the products of lipid peroxidation stimulates PROM2 transcription by a mechanism that involves p38 MAP kinase‐mediated activation of HSF1 and HSF1‐dependent transcription of PROM2. HSF1 inhibitors sensitize a wide variety of resistant cancer cells to drugs that induce ferroptosis. Importantly, the combination of a ferroptosis‐inducing drug and an HSF1 inhibitor causes the cytostasis of established tumors in mice, although neither treatment alone is effective. These data reveal a novel approach for the therapeutic induction of ferroptosis in cancer.

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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. Review of manuscript EMM-2020-13792 -Targeting prominin2 transcription to overcome ferroptosis resistance in cancer by Brown et al. Ferroptosis is a novel cell death modality that has attracted considerable interest as a potential therapeutic option to treat a series of tumour states and defined lineages that challenge current treatment options. Therefore there is a pressing need to translate this into therapeutic options that could efficiently induce this form of cell death for therapeutic benefit. In the present manuscript the authors suggest a potential role for the HNE-p38-HSF1-PROM2 axis in suppressing ferroptosis and that this pathway is amenable to pharmacological intervention and could be leveraged to improve ferroptosis inducing strategies. Having said this in this reviewer opinion the articles has a series of short comings that significantly impact on the authors conclusions and would require attention: 1-The role of HNE in activating p38 is not convincingly demonstrated. For instance, concentrations used here (>20µM) are not at all in the physiological range. More importantly, the level of alkylation seen by the authors (for example as in Fig 1E) argues against any kind of specificity. 2-Additionally, the impact of p38 in ferroptosis is weak and relies mostly on pharmacological data using a single inhibitors (BIRB). Therefore it will be important that the authors provide additional evidence for a role of p38 -for instance by introducing constitutively active variants or generating p38 deficient cell lines. 3-Similarly, a link to HSF1 falls short for the same reason, potentially relevant here is the similar functional groups present in the small molecule used to inhibit HFS1 (KRIBB11) which share a similar NO2 group that can be activated to a potent alkylating agent -similar to the GPX4 inhibitor ML210. The knockdown data is also problematic as this seems to rely on a single siRNA and no rescue control was performed. In figure 3G the effect of RSL3 doesn't seem to be related to a specific action of GPX4 as it is know that selectivity of this drugs is lost at concentration higher than 500nM. Additionaly, given the general toxicity expected by HSF1 loss its not entirely clear if the effect will be due to a specific loss of buffering ferroptosis or simply because the cells are on the verge of dying. 4-Another important consideration would to show that the PROM2 expression, potentially using a doxocycline system to titrate expression to levels similarly to the ones observed upon RSL3 challenge, can rescue the sensitizing effects generated by inhibiting p38 and HSF1. 5-Regarding the in vivo experiments it is not clear if indeed ferroptosis or lipid peroxidation is the major culprit of the effects observed. Critical experiments showing that system Xc-deficient xenografts are more sensitive to manipulation of the p38-HSF1 axis would have been informative as well as assess the impact of ferroptosis inhibitors on the IKE+KRIB11 combination.

Referee #2 (Remarks for Author):
This manuscript by Brown. et al examined the mechanism of induction of promin2 (PROM2) expression, which functions to resist ferroptosis under pro-ferroptotic stress. The results reveal that 4HNE-p38 phosphorylation-HSF1 signaling induces PROM2 transcription, and that pharmacological inhibition of p38 and HSF1 can sensitize cancer cells to ferroptosis. These findings provide a contribution to the mechanism of ferroptosis resistance, with potential implication for the therapy of ferroptosis-resistant cancer. However, I have several comments, in particular i) whether 4HNE is a specific lipid metabolite to induce PROM2, and ii) the mechanism of p38 phosphorylation by 4HNE. 1) Lipid peroxidation is known to generate a variety of lipid oxidation byproducts besides 4-HNE, such as 4HHE, HEL, CRA, 4-ONE, and acrolein etc. Yet, the authors only examined the effects of 4-HNE and MDA, and concluded that 4HNE was a specific lipid metabolite for the PROM2 induction. With that being said, the authors should show whether lipid metabolites other than 4-HNE and MDA may influence p38 phosphorylation and PROM2 induction.
2) The mechanism of increased p38 phosphorylation (p-p38) by 4-HNE remains unclear: Is 4-HNEadducted p38 more prone to become phosphorylated? Could it be that free 4HNE or 4HNEadducted upstream molecule(s) promote p38 phosphorylation? Does unphosphorylated p38 fail to bind to 4-HNE? Please clarify these questions.
3) To confirm the proposed signaling for ferroptosis resistance, the effects of p38 and HSF1 inhibitors need to be studied in cells overexpressing PROM2. 4) Additional control groups need to be included for some experiments: Please show the BIRB treatment group without RSL3 in Figure  Affymetrix ID in each set of cancer data. In the data set, the gene symbol PROM2 includes four Affymetrix IDs. The authors used ID of 1562378_s_at in lung cancer and 234198_at in gastric cancer. When I applied a single Affymetrix ID, the tendency of the mortality was not consistent.
Regarding the breast cancer graph, I tried to reproduce it using the KM plotter data; however, all the Affymetrix IDs of PROM2 failed to reproduce the graph shown in the manuscript. Explain this issue and add detailed description in the method or legend for the reproducibility of the results. The technical data is convincing. No data about animal ethics / in vivo protocol approval is given Referee #3 (Remarks for Author): In their manuscript entitled "Targeting prominin2 transcription to overcome ferroptosis resistance in cancer", Caitlin W. Brown et al. worked on cancer cells and they report that 4HNE stimulates PROM2 transcription via an HSF1-dependant mechanism in the context of ferroptosis resistance. The authors showed that 4HNE directly targeted p38MAPK to increase PROM2 mRNA&protein expression levels. In this study, resistance to GPX4 inhibition was obtained by blocking HSF1, both in vitro and in vivo. The article is nicely written, figures are clear, and results are of interest in the aim of stimulating ferroptosis to tackle cancer proliferation and resistance to anticancer treatments. The authors used several cell lines to study and describe the cellular mechanism, which is an asset. Although most of the cellular players involved in the signaling pathway of this work (HSF1, 4HNE, GPX4, p38, see Fig7) were already described separately as being involved in ferroptosis, the authors managed to link them into a single pathway/model of ferroptosis resistance, providing a strong rationale for using different combination of inhibitors leading to effective cancer therapy, especially in the context of trying to overcome resistance to ferroptosis. -in vivo experiments: Please indicate if the study was approved by an independent ethics committee, together with the approval number -in vivo experiments: please provide the number of mice per group, and how many mice were removed from the study earlier (if applicable) -Figs 6B and 6C: the axis legend "days post-treatment" is confusing, since it may correspond to days of treatment? Were those tumors measured daily after treatment disruption? -The reviewer does not understand the treatment sequence (in vivo experiment): in the schematic 6A, treatments start at day 22, until day 35. This is a 13-day period, and this contrasts with the 8day timescale of Figs 6B and 6C. Please clarify in the text and/or material and methods section. -Please, provide the origin/clones used for immunohistochemistry used on tumors (if different from those used for the western blots) -Page 11, the authors state "Notably, 4HNE staining increased with IKE treatment but decreased with the combined treatment compared to the control" Fig 6D does not support this assertion. Did you mean: "Notably, Prominin2 staining increased with IKE treatment but decreased with the combined treatment compared to the control" Minor remarks: -Page 7: line 16, please replace RSLE by RSL3 -Suppl. Fig 4C: To highlight the absence of specific effect of dasatinib and erlotinib on ferroptosis the authors plotted the RSL3/dasatinib/KRIBB11 and RSL3/dasatinib/KRIBB11 effects. It seems that there is an effect at 5 and 6 uM (respectively, which is close to the decrease in survival obtained at 2µM for BIRB and KRIBB11). The controls with RSL3/dasatinib and RSL3/erlotinib are lacking to conclude on this decrease. -Please, provide information (in the Mat&Meth section) on the experiment aiming at obtaining RSL3 resistant MDAMB cells. What was the IC50 of RSL3 before (MDAMB231S) and after (MDAMB231R)? -Page 10, sentence starting with "Mice were randomized": Please add the condition KRIBB11 alone (50mg/kg) in the sentence, as well as in the schematic Fig 6A  -Fig 7: Nice figure, however no sentence in the text refers to Fig.7. Could you please add a short sentence to recapitulate the underlying mechanism and the original pathway described in this work (in the conclusion section, with reference to fig7)?

EMM-2020-13792 Response to Reviewers' Comments
Reviewer 1 Comment #1: The role of HNE in activating p38 is not convincingly demonstrated. For instance, concentrations used here (>20µM) are not at all in the physiological range. More importantly, the level of alkylation seen by the authors (for example as in Fig 1E) argues against any kind of specificity.
Response: We appreciate the reviewer's concern. However, we note the following: 1) There is existing evidence in the literature that 4-HNE activates p38 (see reference 28); 2) We examined the literature and found that physiological range of 4HNE in serum plasma is reported to be as high as 20µM (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038367/). Ferroptotic cells could be assumed to have much higher levels and as such we consider 25µM to be physiologically relevant; 3) We consider the data in Figure 1E to be significant given the difference between the EtOH group and the 4HNE/RSL3 groups, as well as the nature of the experiment (immunoblotting for 4HNE).
Comment #2: "Additionally, the impact of p38 in ferroptosis is weak and relies mostly on pharmacological data using a single inhibitor (BIRB). Therefore, it will be important that the authors provide additional evidence for a role of p38 -for instance by introducing constitutively active variants or generating p38 deficient cell lines." Response: Although BIRB is used frequently as a specific p38 inhibitor, we have now used another specific p38 inhibitor (SB202190) to address the reviewer's concern and observed the same inhibitory effect on PROM2 mRNA expression. These new data are included in Figure EV2D. We believe that extensive characterization of the role of p38 variants in this pathway is unnecessary given the focus of the manuscript.
Comment #3: A link to HSF1 falls short for the same reason, potentially relevant here is the similar functional groups present in the small molecule used to inhibit HFS1 (KRIBB11) which share a similar NO2 group that can be activated to a potent alkylating agent -similar to the GPX4 inhibitor ML210. The knockdown data is also problematic as this seems to rely on a single siRNA and no rescue control was performed. In figure 3G the effect of RSL3 doesn't seem to be related to a specific action of GPX4 as it is known that selectivity of this drugs is lost at concentration higher than 500nM. Additionally, given the general toxicity expected by HSF1 loss its not entirely clear if the effect will be due to a specific loss of buffering ferroptosis or simply because the cells are on the verge of dying." Response: In response to the reviewer's comment, we note that KRIBB11 is considered to be a highly specific HSF-1 inhibitor that has been used in many studies. In addition, we substantiated the KRIBB11 data by using a pooled siRNA (and not a single siRNA) to knock-down HSF-1 expression with similar consequences. Given that we used a pooled siRNA, it was not possible to do a rescue experiment. To address the valid concern raised about general toxicity, we have 10th Apr 2021 1st Authors' Response to Reviewers performed a rescue experiment with ferrostatin-1 to verify that the cells are dying as a result of ferroptosis and not general toxicity. These new data are included in Figure EV3D. Comment #4: Another important consideration would be to show that the PROM2 expression, potentially using a doxycycline system to titrate expression to levels similarly to the ones observed upon RSL3 challenge, can rescue the sensitizing effects generated by inhibiting p38 and HSF1.
Response: To address this concern, we generated MCF10A cells with elevated levels of PROM2 expression and demonstrated that this increase in PROM2 expression has a significant effect on mitigating the effect of inhibiting p38 and HSF1. These new data are included in Figure EV3C. Comment #5: "Regarding the in vivo experiments it is not clear if indeed ferroptosis or lipid peroxidation is the major culprit of the effects observed. Critical experiments showing that system Xc-deficient xenografts are more sensitive to manipulation of the p38-HSF1 axis would have been informative as well as assess the impact of ferroptosis inhibitors on the IKE+KRIB11 combination." Response: We do not believe that an in vivo experiment with xCT-deficient xenografts would add to the manuscript because the Stockwell group has shown that IKE is a selective and stable inhibitor of system xCT and that it induces ferroptosis (see reference 5). They also reported its efficacy in vivo for inducing ferroptosis and slowing tumor growth (see reference 5). Our data add to these findings by demonstrating that HSF1 inhibition in vivo sensitizes resistant cells to IKE.

Reviewer 2
Comment #1: "Lipid peroxidation is known to generate a variety of lipid oxidation byproducts besides 4-HNE, such as 4HHE, HEL, CRA, 4-ONE, and acrolein etc. Yet, the authors only examined the effects of 4-HNE and MDA, and concluded that 4HNE was a specific lipid metabolite for the PROM2 induction. With that being said, the authors should show whether lipid metabolites other than 4-HNE and MDA may influence p38 phosphorylation and PROM2 induction." Response: In response to this comment, we emphasize that we compared two reactive lipid species that are associated with ferroptosis (4HNE and MDA) and concluded that 4HNE induces PROM2 and that MDA does not. We are not concluding that other lipid reactive species may not induce PROM2 but that PROM2 can be induced by a specific lipid reactive species (4HNE). We consider this finding to be novel and significant. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received feedback from the two referees who re-reviewed your manuscript. As you will see from the reports below, both referees acknowledge your efforts to address their initial concerns, and recognize that the manuscript has significantly improved. However, they also both raise issues that remain unanswered.
As EMBO Press encourages a single round of major revisions only, we would normally reject the manuscript at this stage. However, as both reviewers recognize (as we do) the interest of the study, we would like to exceptionally invite a second round of revisions. Please be aware that this will be the last chance for you to address the points raised by the referees. As indicated below, we do not ask for experimental validation of all the referees' points. More specifically: Referee #1: Comment 1: please address this comment in writing, both in the rebuttal and in the manuscript. Comment #2: if you have data at hand (genetic evidence), we will be happy for you to include it, however experimental data will not be required for further consideration of the manuscript. Comment #3: please address this point in writing, both in the rebuttal and in the manuscript. Comment #4: please include the whole range of concentrations and discuss the use of MCF10A. Comment #5: we do not ask you to provide additional xenograft experiments. Please discuss.
Referee #2: Comment #1: please address this point experimentally. Comment #2: please discuss. Comment #3: please clarify. Please also address the minor points from this referee.
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-Please make sure all figures are referenced in the main text. -As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF.
EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. *Addit ional import ant informat ion regarding figures and illust rat ions can be found at ht tps://bit .ly/EMBOPressFigurePreparat ionGuideline ***** Reviewer's comments ***** Referee #1 (Remarks for Author): Comments to the replies presented for the initial review round of manuscript EMM-2020-13792 First of all I would like to thank the authors for their efforts to reply and try to accommodate the initial concerns/comments raised. We are all aware of the difficulties to approach this during this challenging times. Comment #1: The role of HNE in activating p38 is not convincingly demonstrated. For instance, concentrations used here (>20µM) are not at all in the physiological range. More importantly, the level of alkylation seen by the authors (for example as in Fig 1E) argues against any kind of specificity. Response: We appreciate the reviewer's concern. However, we note the following: 1) There is existing evidence in the literature that 4-HNE activates p38 (see reference 28); 2) We examined the literature and found that physiological range of 4HNE in serum plasma is reported to be as high as 20µM (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038367/). Ferroptotic cells could be assumed to have much higher levels and as such we consider 25µM to be physiologically relevant; 3) We consider the data in Figure 1E to be significant given the difference between the EtOH group and the 4HNE/RSL3 groups, as well as the nature of the experiment (immunoblotting for 4HNE). Reply: The evidence mentioned by the author, mostly the citations within the cited reference, are by no means unequivocal. Measuring 4-HNE levels unambiguously is rather complicated, given the reactive nature of this electrophile. Overall, the 20µM range of free 4-HNE appears to be exaggerated. Having said this, the scope of this discussion potentially falls out of the current work but the manuscript would benefit from highlighting these potential shortcomings. Finally, Figure 1E  ). Yet in that study the notion of lack of specificity is also evident, and furthermore, p38 was not found as a potential target of endogenously generated electrophiles and also given the high. Again, these differences between experimental conditions are complex, but I would also encourage them to tune their discussion based on this recognition. Comment #2: "Additionally, the impact of p38 in ferroptosis is weak and relies mostly on pharmacological data using a single inhibitor (BIRB). Therefore, it will be important that the authors provide additional evidence for a role of p38 -for instance by introducing constitutively active variants or generating p38 deficient cell lines." Response: Although BIRB is used frequently as a specific p38 inhibitor, we have now used another specific p38 inhibitor (SB202190) to address the reviewer's concern and observed the same inhibitory effect on PROM2 mRNA expression. These new data are included in Figure EV2D. We believe that extensive characterization of the role of p38 variants in this pathway is unnecessary given the focus of the manuscript. Reply: The authors provide data with an additional inhibitor, which strengthens their point, yet the lack of genetic evidence makes the link weak. In this reviewer assessment, the role of p38 is not out of the manuscript focus, and the experiments suggested would not have required substantial time or resources. Comment #3: A link to HSF1 falls short for the same reason, potentially relevant here is the similar functional groups present in the small molecule used to inhibit HFS1 (KRIBB11) which share a similar NO2 group that can be activated to a potent alkylating agent -similar to the GPX4 inhibitor ML210. The knockdown data is also problematic as this seems to rely on a single siRNA and no rescue control was performed. In figure 3G the effect of RSL3 doesn't seem to be related to a specific action of GPX4 as it is known that selectivity of this drugs is lost at concentration higher than 500nM. Additionally, given the general toxicity expected by HSF1 loss its not entirely clear if the effect will be due to a specific loss of buffering ferroptosis or simply because the cells are on the verge of dying." Response: In response to the reviewer's comment, we note that KRIBB11 is considered to be a highly specific HSF-1 inhibitor that has been used in many studies. In addition, we substantiated the KRIBB11 data by using a pooled siRNA (and not a single siRNA) to knock-down HSF-1 expression with similar consequences. Given that we used a pooled siRNA, it was not possible to do a rescue experiment. To address the valid concern raised about general toxicity, we have performed a rescue experiment with ferrostatin-1 to verify that the cells are dying as a result of ferroptosis and not general toxicity. These new data are included in Figure EV3D. Reply: Thank you to the authors for clarifying this -to this reviewer understanding, the lack of a rescue experiment undermines these conclusions' strength and should be discussed accordingly. Additionally, the authors might want to acknowledge that the rescue of Fer-1 is only partial and that there might be non-ferroptosis related toxicity/cell death taking place.
Comment #4: Another important consideration would be to show that the PROM2 expression, potentially using a doxycycline system to titrate expression to levels similarly to the ones observed upon RSL3 challenge, can rescue the sensitizing effects generated by inhibiting p38 and HSF1. Response: To address this concern, we generated MCF10A cells with elevated levels of PROM2 expression and demonstrated that this increase in PROM2 expression has a significant effect on mitigating the effect of inhibiting p38 and HSF1. These new data are included in Figure EV3C. Reply: Could the author present the whole range of concentrations as done for the other experiments. This single point experiment indicates that forced expression of PROM2 only partially protects cells from the combination of drugs. Also, the overexpression of PROM2 in the MCF10A cells seems like a bad choice since this cell line's is highly resistant to ferroptosis even when both compounds are combined.
Comment #5: "Regarding the in vivo experiments it is not clear if indeed ferroptosis or lipid peroxidation is the major culprit of the effects observed. Critical experiments showing that system Xc-deficient xenografts are more sensitive to manipulation of the p38-HSF1 axis would have been informative as well as assess the impact of ferroptosis inhibitors on the IKE+KRIB11 combination." Response: We do not believe that an in vivo experiment with xCT-deficient xenografts would add to the manuscript because the Stockwell group has shown that IKE is a selective and stable inhibitor of system xCT and that it induces ferroptosis (see reference 5). They also reported its efficacy in vivo for inducing ferroptosis and slowing tumor growth (see reference 5). Our data add to these findings by demonstrating that HSF1 inhibition in vivo sensitizes resistant cells to IKE. Reply: Here, I would like to point to the authors that in reference 5, IKE was only shown to drive ferroptosis in vitro unequivocally. In vivo and unequivocal evidence is lacking. For instance, there is no experiment showing that lipid peroxidation contributes to decreased tumour growth, which would characterize ferroptosis. Moreover, since it is known that system Xc-is not required for cysteine uptake (in the form of cystine) in vivo, given that Xct knockout mice are fully viable, the question still stands. Does IKE drives cell death by inhibiting system Xc-and drives ferroptosis in vivo? Therefore, despite cumbersome, the xenograft would have helped to answer this question and ultimately provide evidence for this. Ultimately, the same applies here, does the combination of IKE+KRIB11 induces ferroptosis in vivo? This is not to discredit the practical impact of the combination but to highlight that this is not unequivocally shown, and therefore the authors should acknowledge these limitations in their discussion.

Referee #2 (Remarks for Author):
The authors appropriately responded most of the reviewer's comments. However, the following questions remains.
1. Although the finding of PROM2 induction by 4HNE is novel as the authors responded, the authors only examined the effects of two lipid oxidation byproducts. Thus, to examine whether other lipid reactive species (such as 4HHE, HEL, CRA, and acrolein) can induce the p38-HSF1-PROM2 signaling is still recommended. The compounds are commercially available and the additional analysis would not be difficult to do. Figure EV3C, KRIBB1 still enhanced the sensitivity of RSL3 despite the PROM2 overexpression independent of p38-HSF1 signaling. Please explain the reason of this finding. Also, please add the WB data showing overexpression of PROM2 in MCF10A. "Increased expression of prominin2 in MCF10A cells diminished their sensitivity to p38 and HSF1 inhibition ( Figure EV3C)." This sentence is not correct because the authors did not statistically compare the survival rate between mock cells and overexpression cells.

Minor
In Figure EV3B, an unnecessary word of "60" was overlapped. In the legend of figure EV3, "5 mM of RSL3" and "2 mM ferrostatin-1" would be incorrect unit.
I would appreciate your advice in responding to two of the comments of Reviewer #1, which you asked us to address in writing.
Comment #1: "The evidence mentioned by the author, mostly the citations within the cited reference, are by no means unequivocal. Measuring 4-HNE levels unambiguously is rather complicated, given the reactive nature of this electrophile. Overall, the 20µM range of free 4-HNE appears to be exaggerated. Having said this, the scope of this discussion potentially falls out of the current work but the manuscript would benefit from highlighting these potential shortcomings. Finally, Figure 1E  ). Yet in that study the notion of lack of specificity is also evident, and furthermore, p38 was not found as a potential target of endogenously generated electrophiles and also given the high. Again, these differences between experimental conditions are complex, but I would also encourage them to tune their discussion based on this recognition".
The reviewer suggests that the 20uM concentration of 4-HNE may be exaggerated but admits that this issue potentially falls out of the scope of the paper and indicates that is difficult to assess the appropriate concentration. We are unsure how to modify the text.
From our perspective, the data in Fig. 1E demonstrate specificity, especially with the EtOH control. Also, the reviewer states that the differences between experimental conditions are complex but asks us to tune our discussion. Again, we are unsure how to modify the text. Comment # 5: Here, I would like to point to the authors that in reference 5, IKE was only shown to drive ferroptosis in vitro unequivocally. In vivo and unequivocal evidence is lacking. For instance, there is no experiment showing that lipid peroxidation contributes to decreased tumour growth, which would characterize ferroptosis. Moreover, since it is known that system Xc-is not required for cysteine uptake (in the form of cystine) in vivo, given that Xct knockout mice are fully viable, the question still stands. Does IKE drives cell death by inhibiting system Xc-and drives ferroptosis in vivo? Therefore, despite cumbersome, the xenograft would have helped to answer this question and ultimately provide evidence for this. Ultimately, the same applies here, does the combination of IKE+KRIB11 induces ferroptosis in vivo? This is not to discredit the practical impact of the combination but to highlight that this is not unequivocally shown, and therefore the authors should acknowledge these limitations in their discussion.
The Stockwell paper (ref 5) that the reviewer mentions concludes that IKE induces 5 May 2021 Author correspondence ferroptosis in vivo. Although the data may not be as rigorous as the reviewer would like, the data in this paper demonstrate that IKE can inhibit tumor growth in vivo by a mechanism that involves ferroptosis. We add to these findings by demonstrating that tumor that are resistant to IKE can be sensitized by co-treatment with the HSF-1 inhibitor.
We are not sure how to respond with diminishing the impact of the Stockwell paper and our own work.

May 2021
Editor correspondence I have now received the feedback from referee #1, who stated: "regarding comment 1; they lack evidence for an unequivocal and specific role for 4-HNE, so I would suggest that the discussion is modified and the prominent role for 4-HNE be less explicit. One way to approach this would be to justify the effects are derived from lipid peroxidation byproducts such as 4-HNE (but also others!) regarding comment 5; the problem is that triggering ferroptosis in vitro and in vivo is very different, particularly when using system Xc-inhibitors. This is exemplified for instance by the phenotype of system Xc-KO mice, which are fully viable but cells derived from these mice readily die in culture. And the sole reason is, cystine is not a major source for cysteine in vivo, it is in vitro though.
So when the authors say that their drug combination triggers ferroptosis in vivo using a system Xc-inhibitor, which is expected to drive cysteine deprivation I don't see the evidence for this. Currently, there is not enough evidence showing that IKE has an ontarget (Xct) activity in vivo, even it would have its is very likely that in vivo inhibition of system Xc-would not lead to cysteine starvation, simply because this is not how cells acquired cysteine in vivo. I just would like to state again, the same evidence is missing in the paper they cite from the Stockwell group. Yet, its obvious that the IKE is doing something in vivo as reported by both works -but none actually provided evidence that this is via system Xc-or ferroptosis (again, in vivo). Overall for the field, this is something important, otherwise, many more people might be misled to use IKE as an in vivo ferroptosis-inducing agent -leading to pointless waste of mice and money." Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees who re-reviewed your manuscript. As you will see, they are supportive of publication, and I am therefore pleased to inform you that we will be able to accept your manuscript once the following editorial points will be addressed: 1/ Main manuscript text: -Please answer/correct the changes suggested by our data editors in the main manuscript file attached (in track changes mode). Please use this file for any further modification. 3/ We would also encourage you to include the source data for figure panels that show essential data. Numerical data should be provided as individual .xls or .csv files (including a tab describing the data). For blots or microscopy, uncropped images should be submitted (using a zip archive if multiple images need to be supplied for one panel). Additional information on source data and instruction on how to label the files are available at . 4/ Checklist: Please fill in section B/2 and B/5. 5/ Thank you for providing a synopsis text and figure. I slightly edited the text to fit our style and format, please let me know if you agree with the following: Stimulating ferroptosis has emerged as a potential therapeutic strategy against cancer. Some tumor cells, however, are resistant to known ferroptosis stimuli. This study identifies mechanisms that contribute to ferroptosis and develops strategies to overcome resistance. • Prominin 2 expression was induced by ferroptotic stimuli and contributed to resistance. • PROM2 transcription was stimulated by the lipid metabolite 4-hydroxynonenal (4-HNE) via a mechanism involving p38 MAP-kinase-mediated activation of heat shock factor 1 (HSF1). • Resistant tumor cells were sensitized to drugs that induce ferroptosis by HSF1 inhibition.
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