IRE1 RNase controls CD95-mediated cell death

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   Review Commons

   Mar 23, 2023

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   General statement

   Several studies have previously demonstrated functional links between the death receptors (DR) TRAIL-R1/2 and the Unfolded protein response (UPR). In this manuscript, we describe the previously unrecognized IRE1-dependent dual regulation of the expression of another DR, CD95, and CD95L-induced cell death. Our work therefore adds to the current knowledge on the functional links existing between UPR and DR signaling and provides novel mechanistic insights on a dual regulation involving both transcriptional and post-transcriptional control of the expression of CD95 mRNA expression by IRE1. To …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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   Reply to the reviewers

   Point-by-point response to reviewer comments

   General statement

   Several studies have previously demonstrated functional links between the death receptors (DR) TRAIL-R1/2 and the Unfolded protein response (UPR). In this manuscript, we describe the previously unrecognized IRE1-dependent dual regulation of the expression of another DR, CD95, and CD95L-induced cell death. Our work therefore adds to the current knowledge on the functional links existing between UPR and DR signaling and provides novel mechanistic insights on a dual regulation involving both transcriptional and post-transcriptional control of the expression of CD95 mRNA expression by IRE1. To demonstrate this, we have used both genetic (overexpression of XBP1s or dominant-negative forms of IRE1) and pharmacologic (IRE1 RNase inhibitor) approaches and cellular models of glioblastoma (GB) and triple-negative breast cancer (TNBC). We show that IRE1 RNase activity promotes CD95 expression and CD95-mediated cell death via the transcription factor XBP1s whilst IRE1 RNase limits CD95 expression and cell death via its ability to cleave RNAs (through RIDD, for Regulated IRE1-dependent decay of RNAs, activity). Furthermore, we report that IRE1-mediated control of CD95 expression is active in vivo, using a model of CD95-mediated fulminant hepatitis in mice. Lastly, we correlate these results to the pathology by showing that CD95 expression is decreased in RIDD high or XBP1s low human GB and TNBC tumors.

   We thank the reviewers for their fair assessment of our manuscript and for their insightful comments. Below, we describe the experiments we plan to carry out to address the reviewers’ comments.

   Reviewer #1 (Evidence, reproducibility and clarity (Required)): Summary: Here the authors argue that IRE1 activation has opposite effects on Fas/CD95 expression/stability in a number of contexts, via either RIDD-dependent degradation of Fas mRNA or XBP-1-mediated induction of Fas expression, which led to either increased or decreased sensitivity to Fas-induced apoptosis in a number of settings. Major issues: The study is somewhat preliminary and inconclusive in that it is not clear why the RIDD function of XBP-1 appears to predominate in vitro in the cell lines examined, leading to modest increases in Fas expression levels (Figure 1) when IRE1 DN versus IRE1 WT constructs are overexpressed, which is at odds with the latter part of the paper which suggests that inhibition of RIDD led to reduced Fas expression levels. However, this could be due to supraphysiological levels of IRE1 being expressed under overexpression conditions, leading to confounding results. Similarly, when XBP-1s is overexpressed in vitro (Figure 5) the modest increases in CD95/Fas expression and sensitization to Fas-induced cell death may not be fully representative of what would be observed at physiological levels of XBP-1s activation. The in vivo results obtained using an IRE1 RNase inhibitor (MKC8866) contradict the earlier part of the study (as Fas levels decreased and there was protection from Fas-induced liver toxicity) and this could be due to a multitude of reasons. There is no doubt that impacting on IRE1 activity has interesting effects on CD95/Fas expression, which can be up- or -down-regulated, with consequences for cell death induced via engagement of the latter receptor, however, the manuscript does not offer a lot of clarity on which outcome is the predominant one in the context of engagement of the UPR. I have the following suggestions for improvement.

   We thank the reviewer for this overall positive assessment.

   1. The authors should induce ER stress using Thaps, Brefeldin A and Tunicamycin, and explore the effects of doing this on Fas expression levels in the context of silencing endogenous IRE1, XBP-1 and PERK.

   We do agree with this reviewer that the proposed experiments might further highlight which of the IRE1-dependent control of CD95 expression dominates upon ER stress induction. Therefore, we will perform the requested experiment in the various cell lines already used in the manuscript.

   We propose to evaluate the expression of CD95 (at the mRNA and total protein levels) under ER stress induction (by different ER stressors) upon knock-down of IRE1 or XBP1. Other DRs (TRAIL-R1 and 2) have been shown to be induced by PERK activation and it is also demonstrated that PERK and IRE1 signaling pathways coregulate each other. As such, we also propose to assess whether PERK could also control CD95 expression in this setting.

   1. The authors should explore the effects of silencing of IRE1, XBP-1 and PERK on constitutive Fas expression and the outcome of Fas/CD95-induced apoptosis in cells not experiencing an overt activation of the UPR (i.e. in the absence of Thaps, Brefeldin A or other UPR inducer).

   We thank the reviewer for their suggestion and will perform the requested experiments as proposed.

   1. The specificity of MKC8866 at the concentration used (30 uM) is unclear. What effect does MKCC have on sensitivity towards Fas-induced apoptosis, similar to the type of experimental set up presented in Figure 5A, 5B?

   Regarding the specificity of MKC8866, this drug has been optimized and refined from a family of IRE1-specific endoribonuclease inhibitors initially obtained from a chemical library screen [1-3]. This salicylaldehyde analog has already shown to be effective in multiple cancer models including breast [4, 5] and prostate [2] cancers. We have recently demonstrated its efficacy in a GB mouse model [6]. It is therefore a widely used IRE1 inhibitor, including in the dose range 10-30 mM used in this study (e.g [4, 5]). We therefore do not think it is in the scope of this manuscript to re-assess it specificity. However, we will aim at testing an additional IRE1 inhibitor to assess whether similar effects can be observed on CD95 expression in cells. To do so, we propose to use a novel IRE1 kinase inhibitor developed in the laboratory (DOI: 10.26434/chemrxiv-2022-2ld35 – Accepted iScience) and shown to efficiently blunt IRE1 activity in GB. As also suggested by the reviewer, we will assess whether the use of MKC-8866 can affect CD95L-induced cell death in cell lines.

   1. Similarly, what effects does MKC8866 (at 30 uM) have on key Fas pathway determinants, such as Fas, FLIPL, FLIPs, Caspase-8, FADD, RIPK1, A20, CYLD, cIAP-1, cIAP-2 and Bid? There are many points at which MKC8866 could influence the outcome of Fas receptor engagement beyond the receptor itself.

   In the present manuscript, we have shown that MKC-8866 reduces CD95 expression in mouse liver (IHC depicted in Figure 4B and S3B) in vivo and that, when used at 30 mM in vitro, it prevents the loss of CD95 expression induced by tunicamycin or thapsigargin in U87 cells (Fig 1C-F). We do agree with the reviewer that IRE1 may impact CD95-induced cell death beyond modulating CD95 expression, as also already discussed in the present manuscript. Therefore, and as suggested, we will assess whether MKC-8866, used at 30 mM, also impacts on the basal cellular expression of the various components of CD95 signaling mentioned by this reviewer.

   Minor issues:

   1. For the Fas mRNA cleavage experiments presented in Figure 2, there are no irrelevant control mRNAs to allow the reader to judge whether the effects presented are specific to Fas mRNA or are commonly observed for many mRNAs at these amounts of IRE1 (1 ug, 0.5 ug, which appear high).

   The expression of Fas mRNA was already normalized to GAPDH (which does not seem to vary upon incubation with IRE1). We nevertheless will test the expression of additional “irrelevant” RNAs as suggested by the reviewer.

   Reviewer #1 (Significance (Required)): General assessment: this is an interesting study, as there is little knowledge currently concerning how the UPR influences Fas expression or Fas-dependent outcomes. However, the impact of this work is limited by the overexpression approaches used, which could produce artifactual results, as well as the contradictory message of the study.

   Although we think that the message of the manuscript is indeed complex, the work presented herein does not rely exclusively on overexpression approaches as our genetic-based results are also comforted by the use of pharmacologic inhibitors of IRE1.

   Advance: the advance reported here is relatively modest and limited in scope due to the inconclusive nature of the data presented.

   Audience: this study will be of interests to specialists in the UPR and cell death communities.

   We thank the reviewer for acknowledging the overall novelty of our work. We do hope that the experiments proposed will address her/his remaining concerns.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   The authors address here for the first time the connection between CD95, which is known as Fas, and ER stress. The role of another DR, TRAIL-R2 has been already reported, but this is the first study uncovering the link between Cd95 system and ER stress. The study is performed on the high level and supported by all necessary controls. They find the connection between IRE1 and CD95 and show that it might play a role in Cd95 signaling and attenuate CD95-mediated cell death.

   Further, the correlation between CD95 expression and IRE is found in tumors. Importantly the authors find out the connection between XBP1 and CD95 expression, which was not reported to date. Hence, it is a very important and highly essential piece of research.

   We thank the reviewer for these very positive comments and the acknowledging of the novelty and importance of our study.

   However, I would like to clarify the several issues:

   1: Figure 1. Tunicamycin obviously leads to deglycosylation of CD95, which is indicated by the appearance of 35 Kda band. This should be highlighted and commented.

   We agree, this will be commented on in the text.

   1. Figure 2c, d. The piece of mRNA structure, which is synthesized, might have the different secondary structure and might be not cleaved by IRE, accordingly. More detailed comments have to be provided in this regard.

   The model depicted in Figure 2B is a predicted computational secondary structure of CD95 mRNA. In the experiments performed in Figure 2A, C and D the mRNA was extracted from U87 cells prior to incubation with recombinant IRE1 and the resulting products analyzed using RT-qPCR with primers flanking different portions of the CD95 mRNA sequence. For Figures 2C and D, the primers used flank the two sites which were predicted to be cleaved by IRE1 based on previous work from our lab [7]. Even though we cannot exclude that additional sites can be targeted beyond these two, the fact that the amplification of CD95 sequence is reduced in samples pre-incubated with recombinant IRE1 strongly suggests that IRE1 is indeed able to cleave CD95 mRNA in these regions in vitro. We will modify the main text to further explain this point.

   1. Figure 3. Caspase-8-3 western blots show beautiful effects but did authors see some effects further downstream, eg on PARP1 cleavage? Was cell death (not viability) measured as well? Can you comment on this?

   This is absolutely right, we will test PARP-1 cleavage in this setting as suggested. Given the morphology of the cells we observed in the viability experiments, we would expect a similar trend using cell death assays. However, we do agree with the reviewer that this should be proven experimentally, so we will perform these experiments again using cell death assays as a read out.

   1. Did the authors looked at the DISC assembly? Did they detect some differences there?

   No, we did not. We would expect some difference given the impact we have observed on CD95 expression, caspase-8 activation and cell death of expressing dominant negative forms of IRE1, but this of course needs to be actually tested. We are in the process of optimizing CD95 DISC experiments in our lab and we therefore hope to be able to address this reviewer’s comment in a revised version of the manuscript.

   Reviewer #2 (Significance (Required)):

   This is an excellent study. The authors address here for the first time the connection between CD95, which is known as Fas, and ER stress. The role of another DR, TRAIL-R2 has been already reported, but this is the first study uncovering the link between Cd95 system and ER stress. The study is performed on the high level and supported by all necessary controls. This is an important advance for the death receptor field.

   Thank you again for these very positive comments and your insightful appreciation of our work.

   References

   1. Volkmann, K., Lucas, J. L., Vuga, D., Wang, X., Brumm, D., Stiles, C., Kriebel, D., Der-Sarkissian, A., Krishnan, K., Schweitzer, C., Liu, Z., Malyankar, U. M., Chiovitti, D., Canny, M., Durocher, D., Sicheri, F. & Patterson, J. B. (2011) Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease, J Biol Chem. 286, 12743-55.
   2. Sheng, X., Nenseth, H. Z., Qu, S., Kuzu, O. F., Frahnow, T., Simon, L., Greene, S., Zeng, Q., Fazli, L., Rennie, P. S., Mills, I. G., Danielsen, H., Theis, F., Patterson, J. B., Jin, Y. & Saatcioglu, F. (2019) IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling, Nat Commun. 10, 323.
   3. Langlais, T., Pelizzari-Raymundo, D., Mahdizadeh, S. J., Gouault, N., Carreaux, F., Chevet, E., Eriksson, L. A. & Guillory, X. (2021) Structural and molecular bases to IRE1 activity modulation, Biochem J. 478, 2953-2975.
   4. Logue, S. E., McGrath, E. P., Cleary, P., Greene, S., Mnich, K., Almanza, A., Chevet, E., Dwyer, R. M., Oommen, A., Legembre, P., Godey, F., Madden, E. C., Leuzzi, B., Obacz, J., Zeng, Q., Patterson, J. B., Jager, R., Gorman, A. M. & Samali, A. (2018) Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy, Nat Commun. 9, 3267.
   5. Almanza, A., Mnich, K., Blomme, A., Robinson, C. M., Rodriguez-Blanco, G., Kierszniowska, S., McGrath, E. P., Le Gallo, M., Pilalis, E., Swinnen, J. V., Chatziioannou, A., Chevet, E., Gorman, A. M. & Samali, A. (2022) Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer, Nat Commun. 13, 2493.
   6. Le Reste, P. J., Pineau, R., Voutetakis, K., Samal, J., Jégou, G., Lhomond, S., Gorman, A. M., Samali, A., Patterson, J. B., Zeng, Q., Pandit, A., Aubry, M., Soriano, N., Etcheverry, A., Chatziioannou, A., Mosser, J., Avril, T. & Chevet, E. (2020) Local intracerebral Inhibition of IRE1 by MKC8866 sensitizes glioblastoma to irradiation/chemotherapy in vivo, 841296.
   7. Voutetakis, K. D., D.; Vlachavas, E-I., Leonidas, DD.; Chevet, E.; Chatzioannou, A. (In preparation) RNA sequence motif and structure in IRE1-mediated cleavage.

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2. 

   Review Commons

   Mar 23, 2023

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The authors address here for the first time the connection between CD95, which is known as Fas, and ER stress. The role of another DR, TRAIL-R2 has been already reported, but this is the first study uncovering the link between Cd95 system and ER stress.The study is performed on the high level and supported by all necessary controls. They find the connection between IRE1 and CD95 and show that it might play a role in Cd95 signaling and attenuate CD95-mediated cell death.

   Further, the correlation between CD95 expression and IRE is found in tumors. Importantly the authors find out the connection between XBP1 and CD95 expression, which …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The authors address here for the first time the connection between CD95, which is known as Fas, and ER stress. The role of another DR, TRAIL-R2 has been already reported, but this is the first study uncovering the link between Cd95 system and ER stress.The study is performed on the high level and supported by all necessary controls. They find the connection between IRE1 and CD95 and show that it might play a role in Cd95 signaling and attenuate CD95-mediated cell death.

   Further, the correlation between CD95 expression and IRE is found in tumors. Importantly the authors find out the connection between XBP1 and CD95 expression, which was not reported to date. Hence, it is a very important and highly essential piece of research.

   However, I would like to clarify the several issues:

   1: Figure 1. Tunicamycin obviously leads to deglycosylation of CD95, which is indicated by the appearance of 35 Kda band. This should be highlighted and commented. 2.Figure 2c, d. The piece of mRNA structure, which is synthesized, might have the different secondary structure and might be not cleaved by IRE, accordingly. More detailed comments have to be provided in this regard.

   1. Figure 3. Caspase-8-3 western blots show beautiful effects but did authors see some effects further downstream, eg on PARP1 cleavage? Was cell death ( not viability) measured as well? Can you comment on this?
   2. Did the authors looked at the DISC assembly? Did they detect some differences there?

   Significance

   This is an excellent study. The authors address here for the first time the connection between CD95, which is known as Fas, and ER stress. The role of another DR, TRAIL-R2 has been already reported, but this is the first study uncovering the link between Cd95 system and ER stress.The study is performed on the high level and supported by all necessary controls. This is an important advance for the death receptor field.

   Read the original source
3. 

   Review Commons

   Mar 23, 2023

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Here the authors argue that IRE1 activation has opposite effects on Fas/CD95 expression/stability in a number of contexts, via either RIDD-dependent degradation of Fas mRNA or XBP-1-mediated induction of Fas expression, which led to either increased or decreased sensitivity to Fas-induced apoptosis in a number of settings.

   Major issues:

   The study is somewhat preliminary and inconclusive in that it is not clear why the RIDD function of XBP-1 appears to predominate in vitro in the cell lines examined, leading to modest increases in Fas expression levels (Figure 1) when IRE1 DN versus IRE1 WT constructs are overexpressed, …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Here the authors argue that IRE1 activation has opposite effects on Fas/CD95 expression/stability in a number of contexts, via either RIDD-dependent degradation of Fas mRNA or XBP-1-mediated induction of Fas expression, which led to either increased or decreased sensitivity to Fas-induced apoptosis in a number of settings.

   Major issues:

   The study is somewhat preliminary and inconclusive in that it is not clear why the RIDD function of XBP-1 appears to predominate in vitro in the cell lines examined, leading to modest increases in Fas expression levels (Figure 1) when IRE1 DN versus IRE1 WT constructs are overexpressed, which is at odds with the latter part of the paper which suggests that inhibition of RIDD led to reduced Fas expression levels. However, this could be due to supraphysiological levels of IRE1 being expressed under overexpression conditions, leading to confounding results. Similarly, when XBP-1s is overexpressed in vitro (Figure 5) the modest increases in CD95/Fas expression and sensitization to Fas-induced cell death may not be fully representative of what would be observed at physiological levels of XBP-1s activation. The in vivo results obtained using an IRE1 RNase inhibitor (MKC8866) contradict the earlier part of the study (as Fas levels decreased and there was protection from Fas-induced liver toxicity) and this could be due to a multitude of reasons. There is no doubt that impacting on IRE1 activity has interesting effects on CD95/Fas expression, which can be up- or -down-regulated, with consequences for cell death induced via engagement of the latter receptor, however, the manuscript does not offer a lot of clarity on which outcome is the predominant one in the context of engagement of the UPR. I have the following suggestions for improvement.

   1. The authors should induce ER stress using Thaps, Brefeldin A and Tunicamycin, and explore the effects of doing this on Fas expression levels in the context of silencing endogenous IRE1, XBP-1 and PERK.
   2. The authors should explore the effects of silencing of IRE1, XBP-1 and PERK on constitutive Fas expression and the outcome of Fas/CD95-induced apoptosis in cells not experiencing an overt activation of the UPR (i.e. in the absence of Thaps, Brefeldin A or other UPR inducer).
   3. The specificity of MKC8866 at the concentration used (30 uM) is unclear. What effect does MKCC have on sensitivity towards Fas-induced apoptosis, similar to the type of experimental set up presented in Figure 5A, 5B?
   4. Similarly, what effects does MKC8866 (at 30 uM) have on key Fas pathway determinants, such as Fas, FLIPL, FLIPs, Caspase-8, FADD, RIPK1, A20, CYLD, cIAP-1, cIAP-2 and Bid? There are many points at which MKC8866 could influence the outcome of Fas receptor engagement beyond the receptor itself

   Minor issues:

   1. For the Fas mRNA cleavage experiments presented in Figure 2, there are no irrelevant control mRNAs to allow the reader to judge whether the effects presented are specific to Fas mRNA or are commonly observed for many mRNAs at these amounts of IRE1 (1 ug, 0.5 ug, which appear high).

   Significance

   General assessment: this is an interesting study, as there is little knowledge currently concerning how the UPR influences Fas expression or Fas-dependent outcomes. However, the impact of this work is limited by the overexpression approaches used, which could produce artifactual results, as well as the contradictory message of the study.

   Advance: the advance reported here is relatively modest and limited in scope due to the inconclusive nature of the data presented.

   Audience: this study will be of interests to specialists in the UPR and cell death communities.

   Read the original source
4. 

   Version published to 10.1101/2022.02.25.481813v2 on bioRxiv

   Dec 15, 2022
5. 

   Version published to 10.1101/2022.02.25.481813v1 on bioRxiv

   Feb 26, 2022