IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma
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Abstract
Background
Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils.
Methods
We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings.
Results
We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors.
Conclusions
Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.
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Reply to the reviewers
Rebuttal Preprint RC-2020-00156_
We thank the editor for handling our manuscript and both reviewers for their constructive critiques. We provide below a detailed list of results already available and experiments we propose to perform to address the reviewers’ comments and improve the quality of our manuscript.
Reviewer #1
In this manuscript, Obacz et al. investigated the role of IRE1 signaling in regulating the recruitment of myeloid cells in glioblastoma multiforme (GBM) microenvironment. They show that inhibition of IRE1 signaling decreased polynuclear neutrophil (PN) infiltration to GBM tumors in an animal model; conversely, IRE1 activation correlated …
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
Rebuttal Preprint RC-2020-00156_
We thank the editor for handling our manuscript and both reviewers for their constructive critiques. We provide below a detailed list of results already available and experiments we propose to perform to address the reviewers’ comments and improve the quality of our manuscript.
Reviewer #1
In this manuscript, Obacz et al. investigated the role of IRE1 signaling in regulating the recruitment of myeloid cells in glioblastoma multiforme (GBM) microenvironment. They show that inhibition of IRE1 signaling decreased polynuclear neutrophil (PN) infiltration to GBM tumors in an animal model; conversely, IRE1 activation correlated with higher expression of myeloid cells-attracting chemokines in GBM. They also show that IRE1-XBP1s pathway promotes proinflammatory chemokines in GBM tumor cells through upregulation of UBE2D3, which leads to degradation of the NFκB inhibitor IκB and activation of NFκB downstream signaling. Their finding of a novel IRE1/XBP1s/UBE2D3/NFκB axis is important for understanding the basis of pro-tumoral inflammation in GBM, potentially in other 'immune hot' cancers. The manuscript is well written and the conclusion is well supported by the experiments. However, there are a few critical points that need to be addressed to strengthen their study.
We thank this reviewer for his/her positive comments on our work and for the suggestions made to improve its relevance
Review#1 point 1: In this study, the authors used the GBM primary cell line RADH87 with stable overexpression of wild-type (WT) IRE1 or a truncated IRE1 variant. The expression of wild-type IRE1 was confirmed by Western analysis (Figure S1D). However, the expression of truncated IRE1 variant was not shown.
Response 1.1. The expression on truncated IRE1 variant (designated as Q780* - 80 KDa) is shown in Fig.S1D, following the expression on wild-type (WT) IRE1 (110 KDa). This point will be indicated in the revised version of the supplemental figure legends.
In addition, without tunicamycin treatment, there was no visible difference in XBP1s expression between the cells expressing WT or the mutant IRE1.
__Response 1.2. __Under basal condition, XBP1 splicing is indeed limited and therefore, there is no detectable difference in XBP1s expression level between IRE1 WT and Q780*. In contrast, under tunicamycin treatment (acute stress), reduced XBP1 mRNA splicing is observed (Fig.S1D) thus confirming the functionality of the Q780* truncated form. Of note, RNAseq was performed on these cell lines and basal splicing was quantified showing that even though it is an event that occurs at low frequency, it is decreased in cells expressing the Q780* mutant (this information will be added in the revised manuscript, data are available and analyses ongoing).
*In the Boyden chamber assay (Figure 1C, D), conditioned medium from these cells were used; it was not described whether the cells were treated (e.g. with tunicamycin) to activate the IRE1 pathway. ** *__Response 1.3. __Cells were not treated with Tunicamycin to excluded the impact of other UPR arms in the induction of cytokines expression/myeloid cells attraction. As a consequence, it is the basal secretome (found in conditioned media) that was used in those experiments to evaluate cell migration. We have now strong evidences that blunting IRE1 signaling (either genetically or pharmacologically) has a strong impact on GBM cells proteome and in particular on their secretome even if under basal conditions (manuscript in preparation). This information together with the fact that basal XBP1 mRNA splicing is reduced in IRE1 signaling deficient (Q780* expressing) cells, indicate that in GBM cells, constitutive IRE1 activity contributes to modulate the composition of their secretome towards chemoattraction of myeloid cells. __This point will be further detailed in the results and discussion sections of the revised manuscript. __
Review#1 point 2: The evidence that the mRNA expression of UBE2D3 positively correlates with IRE1/XBP1s pathway is weak. First, In Figure 3D, the correlation between the mRNA expression of UBE2D3 and XBP1 does not seem strong. In addition, as XBP1 mRNA level does not reflect IRE1 activation (as opposed to that of XBP1s), the level of XBP1s instead of total XBP1 should be assessed. Furthermore, such correlation should be validated in additional GBM cohorts/datasets.
Response 2. We agree that the correlation between UBE2D3 and XBP1 mRNA levels in TCGA GBM cohort might not be strong. However data presented in Fig3D were significant. Values indicated in green were Pearson’s correlation values (r). This point will be included in the revised figure legends. Moreover, in the revised version of the manuscript we propose to directly correlate the levels of XBP1s mRNA with the expression levels of __SYVN1, UBE2D3 and UBE2J1 mRNAs. __These data are available from the RNAseq data obtained from the TCGA cohort and already used previously by us (Lhomond et al. Embo Mol Med 2018). In addition, following this observation we have carried out a number of experimental validations using both established and primary GBM cell lines with genetic modifications of XBP1/XBP1s expression as well as ER stress-dependent induction of XBP1s and we clearly demonstrated that XBP1s mRNA levels correlate with UBE2D3 mRNA expression levels (Fig.3G-H, Fig.S2D-E). In addition, in Fig3E using our IRE1 activity signature we have shown a strong correlation between UBE2D3 and XBP1s, which is even more robust than simply correlating the mRNA levels. __Data are already available and analyses are ongoing. __
Review#1 point 3: The results in Figure 3 indicated that XBP1s acts as a transcriptional regulator of UBE2D3 expression. However, it is not clear whether this effect in GBM cells is direct or indirect. Further experiments such as chromatin immunoprecipitation and reporter assays are required to clarify this point.
__Response 3. __We agree with this reviewer’s point. Although we have scrutinized the publicly available ChIPseq databases and found UB2D3 among potential XBP1-regulated genes, we did not validate this observation in our model. To address this point __we propose to perform ChIP experiments __in cells overexpressing a tagged form of XBP1s and validate the presence of UBE2D3 promoter fragments in our experimental system. Moreover, these experiments will also be carried out with endogenous XBP1s (in-house XBP1s antibodies Pluquet et al. Cancer Res. 2013) in our primary GBM lines under basal and ER stress conditions. At last, to further document this, luciferase reporter assays using the UBE2D3 promoter (whose length would be defined based on ChIP experiments and the presence of XBP1s binding sites) upstream the luciferase ORF could be performed. Both ChIP and reporter assays have to be performed.
Review#1 point 4: In addition to UBE2D3, the two other ubiquitin-protein ligases, SYVN1 and UBE2J1, may also be implicated in the degradation of IκB. Did the authors assess their potential role on IκB degradation in their model system?
Response 4. We thank this reviewer for this suggestion. We have previously tested the impact of SYVN1 on IkB degradation with results showing a lot of variation. Indeed even though the trend of our results indicated that SYVN1 silencing appeared to lead to a slight increase in IkB expression, they never reached statistical significance. Variability in the results might be due to the efficacy of SYVN1 silencing and as such we propose to repeat further these experiments with SYVN1 siRNA smart pools to improve silencing efficacy. Moreover, SYVN1 has been shown to also contribute to the ubiquitylation and degradation of IRE1 (Gao et al. Embo Rep 2008; Sun et al. Nat Cell Biol 2015) and has its expression regulated by IRE1 activity (Dibdiakova et al. Neurol Res 2019), it might represent as well a very interesting target to study. Regarding UBE2J1, the situation is less documented. However, it was shown that this E2 works together with SYVN1 in conserved manner to contribute to ERAD (Chen et al. Nat Plants 2016). As such it might also be interesting to test whether the silencing of UBE2J1 impacts on IkB expression. To sum up, we propose to test experimentally whether the silencing of UBE2J1 or SYVN1 or both together impacts on IkB expression (we need to perform the experiments).
Review#1 point 5: *The authors only used ectopic expression of relevant proteins to test their hypothesis in U87 and RADH87 cells. It is necessary to validate these findings using siRNAs/inhibitors for IRE1 and UBE2D3 in a GBM cell line that expresses high levels of endogenous IRE1 and UBE2D3. *
__Response 5. __We propose to test the effect of SYVN1 and UBE2J1 silencing on IkB expression in U87 and RADH87 cells in the revised version of the manuscript (see above). In addition to address this reviewer’s comment, we propose to use __U87 and RADH87 cells overexpressing IRE1 __(Lhomond et al. 2018) and treat them with MKC886, or with siUBE2D3 or with both and evaluate whether in those conditions the NFkB pathway is affected. These experiments should be carried out relatively easily provided that all the recombinant cell lines, drugs and siRNA are already available.
Review#1 point 6: In Figure 3I: The protein expression of UBE2D3 should be shown.
Response 6: We had included control experiments with UBE2B3 expression in FigS3B in the initial version of the manuscript. We will include UBE2D3 expression for Fig3I in the revised version of the manuscript (these data are already available).
Review#1 point 7: In the right panel of Figure 3I: What do the labels #1, 2, 5 mean? Clear descriptions should be provided in the figure legend.
__Response 7. __Those labels correspond to different RADH87 cell lines stably overexpressing UBE2D3 protein. The validation of UBE2D3 expression using Western blotting will be included in FigS3B of the revised version of the manuscript (data are already available).
Review#1 point 8: *In Figure S1D: The expression levels of the truncated IRE1 variant should be shown. *
__Response 8. __The expression on truncated IRE1 variant (designated as Q780*) is shown in Fig.S1D, following the expression on wild-type (WT) IRE1. This point will be indicated in the revised version of the supplemental figure legends.
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Reviewer #2
In the current study, the authors generate evidence supporting a novel pathway downstream of IRE1α/XBP1s in GBM cells involving the activation of an E2-ubiquitin ligase, UBE2D3. In order to do this, they use a combination of patient derived and established cell lines engineered to overexpress IRE1 mutants, XBP1s or UBE2D3. They claim that UBE2D3 is upregulated downstream of XBP1s in GBM cells, and functions to activate NF-kB through the degradation of IkB, thus promoting CXCL2/IL-6/IL-8 production and the subsequent recruitment of monocytes and polymorphonuclear (PN) cells to the tumor microenvironment. However, the article has major shortcomings that need to be addressed before considering its publication
We thank this reviewer for his/her constructive comments on our work.
Review#2 point 1: Fig. 1: Classification of immune cells infiltrating GBM. The characterization of immune infiltrate in GBM is too simplistic. Monocytes, monocyte-derived macrophages and microglia are treated as equivalents along the text (IBA1+), making the story hard to follow. At least in mice, these populations can be easily distinguished based on CD45/CD11b/Ly6C expression (see for example Zhihong Chen et al., Cancer Research, 2017). Can the authors further analyze which of those population are actually affected under IRE1 deficiency and/or UBE2D3 overexpression? On the other hand, it is rather questionable that all CD11b negative cells are exclusively T cells, as suggested in Fig 1B. Can the authors provide evidence and/or references to support their gating strategies?
__Response 1: __We thank the reviewer for this comment. Our objective was to test the impact of IRE1 modulation on the infiltration of myeloid cells in the tumor, and we did not plan to describe this effect on the complete and detailed infiltrating myeloid populations in GBM which could represent a full study on its own. However, to address this reviewer’s critique we propose to complete the characterization of the myeloid population in our mouse model using IHC by adding Ly6C staining for macrophages and granulocytes. We did not select flow cytometry approach to explore this point as suggested by the reviewer (Cheng, Cancer Res, 2017), but instead IHC was preferred as we thought that the localization of the infiltrated immune cells was important to evaluate (periphery vs. core of the tumor). The information about the localization of immune cells is already available and will be added to the revised manuscript. Concerning the second point raised by this reviewer, the strategy to characterize the immune population in human GBM specimen was to combine CD45 and CD11b markers as previously described by Hussain et al. Neuro-Oncol 2006 and Parney et al. J Neurosurg 2009. Moreover, the analysis of additional markers allowed us to confirm that CD45+ CD11b+ cells were mainly monocytic cells (that also co-expressed CD14, CD168, CD64 and HLA-DR); CD45 low CD11b high cells were granulocytes (CD66B, CD15 and CD16); and CD45 high CD11b low cells were mainly CD3+ T cells. These data are already available and will be added to the revised manuscript.
__Review#2 point 2: __*Fig. 1: RADH IRE1 Q780* model - Can the authors further validate the IRE1 deficiency of their model cell line RADH87 IRE1Q780*? It appears to have severely reduced IRE1 levels when compared to the RAD87-IRE1WT cell line (figS1D). Furthermore, the WT and not the truncated form seems to be predominantly expressed. Intriguingly, XBP1 is still being spliced after tunicamycin treatment in this mutant line. All these results differ significantly from the U87-Q780* cell line originally published by Lhomond et al., 2018. Can the authors comment on these differences? Was there a mixture in cell lines? *
__Response 2: __We agree with the reviewer that the level of IRE1Q780* expression on RADH87 cells is lower than the IRE1WT expression (Fig.S1D). As observed by this reviewer, XBP1 was still spliced in Q780* cells but XBP1s expression was reduced as shown in Figure S1D. This is mostly due to the ratio between the expression endogenous IRE1 and that of Q780*, which as previously shown (Lhomond et al; 2018) acts as a dominant negative and preempts endogenous IRE1 signaling. The differences observed are also probably due to the methods used, indeed we measured XBP1 and XBP1s mRNA expression in U87 cells (Lhomond et al. 2018), whereas XBP1s protein expression was used with RADH87 cells (introducing the RNA translation parameter that was not monitored in U87 cells). Differences could be also linked to the cell lines as we used the U87 immortalized and RADH87 primary cell lines.
__Review#2 point 3: __Fig. 1: Impact of IRE1 inhibition on recruitment of myeloid cells to the TME. The experiment in figure 1E-F, which is the only in vivo evidence supporting a role of IRE1 signaling on myeloid cell recruitment, is very hard to interpret. The authors show no evidence that IRE1 is being inhibited under the treatment and if so, up to which extent. Furthermore, what are the cells targeted by MKC in this setting? The differences in the infiltration of PN cells seem very slight, nothing is mentioned regarding the number of mice per group, or the statistical analysis performed. I would suggest performing a simpler experiment to demonstrate an intrinsic effect of IRE1 signaling in GBM cells, comparing the recruitment of myeloid cells in tumors generated by GL261 cells expressing WT vs deficient forms of IRE1.
__Response 3: __The mouse model used in the paper is fully described in (Le Reste BioRxiv 2020 - doi: https://doi.org/10.1101/841296) and all the details about the procedures can be found in this manuscript. This model was developed to recapitulate in mice the standard of care for GBM patient including surgical resection. In addition, drug delivery in brain tumors is often an issue due to the blood-brain barrier. Therefore, the IRE1 inhibitor was delivered locally after resection of the tumor, exposing both tumor and stromal cells. To quantify the myeloid cell recruitment in Fig1E-F, at least thirty random fields from tumor tissue and at least thirty random fields from tumor periphery were quantified for control (PLUG) and MKC-treated group (2 mice/group). The number of positive cells in tumor tissue and tumor periphery were then pulled together for statistical analyses. The significance of the differences in myeloid cells recruitment between control (PLUG) and MKC-treated group was estimated using unpaired student t-test. At least 8 tumors of each group were analyzed comprising 2 to 3 sections of each and 10 fields per section. In addition, we have also performed the experiments using GL261 cells knockout for IRE1, the data are already available and could be possibly added to the revised manuscript.
__Review#2 point 4: __Fig. 2: Correlation between IRE1 signature and cytokine/chemokine signature. In the IRE1 signature as determined in the EMBO Mol Med paper (and to which the authors continuously refer) 6 out of 38 (15%) of the genes correspond to cytokines and/or chemokines (Il6, Il1b, Cxcl2, Cxcl5 and Ccl20) (Lhomond et al., 2018). Besides the fact that it is very unclear how this signature was obtained in the first place, it is rather surprising that in the current paper the authors correlate this "IRE1 activity" signature with the same or other cytokines/chemokines mRNA levels and come to the conclusion that there is a high correlation (fig 2A). Isn't this to be expected? Can the authors clearly explain how the IRE1 signature was determined and prove that their "IRE1 signature" is, in fact, representing IRE1 activity? For instance, it is important to cross validate their results by using an independent signature of IRE1 activity (e.g. ChipSeq XBP1s targets, Chen et al., 2014)?
__Response 4: __We thank this reviewer for asking for precisions about the procedure. The IRE1 signature was fully described in Lhomond et al. 2018 and was obtained from transcriptome datasets obtained from U87 modified for IRE1 activity (Pluquet et al., 2013). IRE1 was validated on GBM patients and appeared as an important tool to evaluate IRE1 activity in tumor specimen not only in GBM but also in other tumor types (Rubio-Patiño C, Cell Metab 2018). Furthermore, IRE1 activity was also directly linked to the pro-inflammatory tumor cell secretome in various studies such as Logue et al. 2018. As indicated by this, some cytokines/chemokines studied in this work were indeed part of the IRE1 signature and correlation between this signature and their expression was indeed expected. However the other main cytokines/chemokines studied here were not present in the IRE1 signature indicating that IRE1 could have been involved in the regulation of their expression. As proposed by reviewer#2, we will include in the revised version of the manuscript the analysis of cytokines/chemokines from the dataset ChipSeq XBP1s targets (Chen et al. 2014), although this study was performed on breast tumors.
__Review#2 point 5: __*Fig 2: XBP1s controlling cytokines/chemokines expression in GBM cells - As suggested by the data on fig1C-D and fig2E, IRE1 appears to be constitutively active in GBM cells, as IRE1 deficiency is sufficient to cause a defect in chemokine production. However, as shown in fig S1D, XBP1s protein was not detected under basal conditions, suggesting that the deficiency in chemokine production in IRE1-deficient cell lines is XBP1s-independent. Can the authors further discuss these results? *
__Response 5: __We thank the reviewer for commenting this point. We think that indeed IRE1 is constitutively active in GBM cells. As we have tested XBP1s protein expression in untreated and tunicamycin-treated RAD87 cells (FigS1D), and we will also provide real time qPCR data to show the presence of basal XBP1s mRNA levels (data already available). We agree that the way we presented the results are misleading and undermine the basal expression of XBP1s. This will be fixed in the revised manuscript.
__Review#2 point 6: __Fig 3: IRE1/XBP1s/UBE2D3/NF-kB axis - Authors must show the activation status of NF-kB in parental U87 cells (Fig3A), as this is a critical evidence to support that IRE1a-deficient U87-DN cells are defective in chemokine production due to an impairment in NF-kB signaling. In addition, even when tunicamycin treatment induce XBP1s and UBE2D3 (figS2D) it does not induce IkB degradation nor NF-kB phosphorylation in parental U87 and RADH87 cells (figS3C) as one should expect if IRE1/XBP1s/UBE2D3/NF-kB pathway is operating in these cells. How can this be explained? Only after XBP1s or UBE2D3 overexpression, NF-kB signaling appears to be affected.
__Response 6: __As shown in Fig3A, U87 cells deficient for IRE1 signaling (DN) exhibit decreased NFkB signaling as exemplified by decreased phospho-NFkB and phospho-IkB compared to control U87 cells proficient for IRE1 signaling. In our manuscript, we mainly focused on the activation of the IRE1/XBP1s/UBE2D3/NFkB signaling axis under basal condition. One could speculate that tunicamycin treatment leads to a strong stress response that others mechanisms are activated that overwhelm the IRE1/XBP1s/UBE2D3 pathway we are describing herein. For instance, it has been demonstrated that the IRE1/JNK signaling was linked to NFkB activation upon acute ER stress (Tam et al. PLoS One. 2012;7(10):e45078; Schmitz et al. Biomedicines. 2018 Jun; 6(2): 58.) and furthermore PERK activation upon thapsigargin or tunicamycin treatment was also found to promote NFkB activation (Deng et al. DOI: 10.1128/MCB.24.23.10161-10168.2004; Fan et al. Cell Death Discov. 2018 Feb 12;4:15). We believe that the pathway we describe here might be linked to constitutive activation of IRE1 signaling (proper to tumor cells) rather than acute activation of this pathway and be compatible with sustained proliferation. To further document this point, we __have already generated data about the phosphorylation status of NFKB in GL261 cells KO for IRE1 compared to the parental cells __(data will be provided in the revised version of the manuscript). In addition we are currently investigating the correlation between IRE1 activity signature and that of NFkB as defined previously (Jin et al. Cancer Res. 2014 May 15; 74(10): 2763–2772.), results should be available shortly and will be added in the revised manuscript.
__Review#2 point 7: __*Fig 4: UBE2D3 and MIB1 – The authors should discuss better what is the possible interaction between UBE2D3 and MIB1. As shown in fig4G, silencing of MIB1 cause a severe increase in UBE2D3 protein levels but this is not commented in the text. *
Response 7: We thank the reviewer for this comment. We believe that MIB1 might also controls the expression of UBE2D3. The data are already available and will be included in the revised version of the manuscript.
__Review#2 point 8: __Fig 6: Chemokines driving recruitment of myeloid cells to UBE2D3 overexpressing tumors. A formal demonstration that GL261-UBE2D3 tumors recruit higher numbers of MM and PNs through an enhanced production of CXCL2, IL-6 and/or IL-8 is lacking. For instance, they could compare the infiltration of myeloid cells in GL261-UBE2D3 vs GL261-UBE2D3-CXCL2KO tumors.
Response 8: To address this point, we propose to test the expression of these cytokines/chemokines in the GL261 tumors after resection using ELISA. These experiments could be carried out in IRE1 KO tumors, in UBE2D3 overexpressing tumors and performed for instance using perfusion of CXCL2, IL6 or IL8 neutralizing antibodies or cells KO for these chemokines. These experiments could be performed but might lead to inconclusive results (not statistically significant) if there is redundancy between the roles of those chemokines. As such, we think that we could provide in vitro information about the respective roles of these chemokines in recruiting MM and PNs but that at present stage the in vivo demonstration is to premature.
__Review#2 point 9: __Authors must provide replicates of the blots to sustain their claims: FigS1D, Fig3A, Fig3I, Fig4G.
Response 9: Replicates and quantifications are already available and will be provided in the revised version of the manuscript.
__Review#2 point 10: __The authors should include a better description of the methods regarding bioinformatic analysis. For instance, which genes where used for MM/PN/T cell signatures in fig1A/S1A?.
Response 10: We thank the reviewer#2. This information is available and a complete description will be included in the revised version of the manuscript.
Review#2 point 11*: *Missing statistical significance on fig 2C and fig 6A to support their claims.
Response 11: Statistical values will be included in the revised manuscript.
Review#2 point 12*: *Fig2F is presented in the text as mRNA levels but in the figure as protein levels.
Response 12: This point will be fixed in the revised version of the manuscript.
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Referee #2
Evidence, reproducibility and clarity
Summary: your understanding of the study and its conclusions.
In the current study, the authors generate evidence supporting a novel pathway downstream of IRE1α/XBP1s in GBM cells involving the activation of an E2-ubiquitin ligase, UBE2D3. In order to do this, they use a combination of patient derived and established cell lines engineered to overexpress IRE1 mutants, XBP1s or UBE2D3. They claim that UBE2D3 is upregulated downstream of XBP1s in GBM cells, and functions to activate NF-kB through the degradation of IkB, thus promoting CXCL2/IL-6/IL-8 production and the subsequent recruitment of monocytes and polymorphonuclear (PN) …
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
Summary: your understanding of the study and its conclusions.
In the current study, the authors generate evidence supporting a novel pathway downstream of IRE1α/XBP1s in GBM cells involving the activation of an E2-ubiquitin ligase, UBE2D3. In order to do this, they use a combination of patient derived and established cell lines engineered to overexpress IRE1 mutants, XBP1s or UBE2D3. They claim that UBE2D3 is upregulated downstream of XBP1s in GBM cells, and functions to activate NF-kB through the degradation of IkB, thus promoting CXCL2/IL-6/IL-8 production and the subsequent recruitment of monocytes and polymorphonuclear (PN) cells to the tumor microenvironment. However, the article has major shortcomings that need to be addressed before considering its publication
Major comments: major issues affecting the conclusions.
-Fig. 1: Classification of immune cells infiltrating GBM The characterization of immune infiltrate in GBM is too simplistic. Monocytes, monocyte-derived macrophages and microglia are treated as equivalents along the text (IBA1+), making the story hard to follow. At least in mice, these populations can be easily distinguished based on CD45/CD11b/Ly6C expression (see for example Zhihong Chen et al., Cancer Research, 2017). Can the authors further analyze which of those population are actually affected under IRE1 deficiency and/or UBE2D3 overexpression? On the other hand, it is rather questionable that all CD11b negative cells are exclusively T cells, as suggested in Fig 1B. Can the authors provide evidence and/or references to support their gating strategies?
-Fig. 1: RADH IRE1 Q780* model Can the authors further validate the IRE1 deficiency of their model cell line RADH87 IRE1Q780? It appears to have severely reduced IRE1 levels when compared to the RAD87-IRE1WT cell line (figS1D). Furthermore, the WT and not the truncated form seems to be predominantly expressed. Intriguingly, XBP1 is still being spliced after tunicamycin treatment in this mutant line. All these results differ significantly from the U87-Q780 cell line originally published by Lhomond et al., 2018. Can the authors comment on these differences? Was there a mixture in cell lines?
-Fig. 1: Impact of IRE1 inhibition on recruitment of myeloid cells to the TME. The experiment in figure 1E-F, which is the only in vivo evidence supporting a role of IRE1 signaling on myeloid cell recruitment, is very hard to interpret. The authors show no evidence that IRE1 is being inhibited under the treatment and if so, up to which extent. Furthermore, what are the cells targeted by MKC in this setting? The differences in the infiltration of PN cells seem very slight, nothing is mentioned regarding the number of mice per group, or the statistical analysis performed. I would suggest performing a simpler experiment to demonstrate an intrinsic effect of IRE1 signaling in GBM cells, comparing the recruitment of myeloid cells in tumors generated by GL261 cells expressing WT vs deficient forms of IRE1.
-Fig. 2: Correlation between IRE1 signature and cytokine/chemokine signature In the IRE1 signature as determined in the EMBO Mol Med paper (and to which the authors continuously refer) 6 out of 38 (15%) of the genes correspond to cytokines and/or chemokines(Il6, Il1b, Cxcl2, Cxcl5 and Ccl20) (Lhomond et al., 2018). Besides the fact that it is very unclear how this signature was obtained in the first place, it is rather surprising that in the current paper the authors correlate this "IRE1 activity" signature with the same or other cytokines/chemokines mRNA levels and come to the conclusion that there is a high correlation(fig 2A). Isn't this to be expected? Can the authors clearly explain how the IRE1 signature was determined and prove that their "IRE1 signature" is, in fact, representing IRE1 activity? For instance, it is important to cross validate their results by using an independent signature of IRE1 activity (e.g. ChipSeq XBP1s targets, Chen et al., 2014)?
-Fig 2: XBP1s controlling cytokines/chemokines expression in GBM cells As suggested by the data on fig1C-D and fig2E, IRE1 appears to be constitutively active in GBM cells, as IRE1 deficiency is sufficient to cause a defect in chemokine production. However, as shown in fig S1D, XBP1s protein was not detected under basal conditions, suggesting that the deficiency in chemokine production in IRE1-deficient cell lines is XBP1s-independent. Can the authors further discuss these results?
-Fig 3: IRE1/XBP1s/UBE2D3/NF-kB axis Authors must show the activation status of NF-kB in parental U87 cells (Fig3A), as this is a critical evidence to support that IRE1a-deficient U87-DN cells are defective in chemokine production due to an impairment in NF-kB signaling. In addition, even when tunicamycin treatment induce XBP1s and UBE2D3 (figS2D) it does not induce IkB degradation nor NF-kB phosphorylation in parental U87 and RADH87 cells (figS3C) as one should expect if IRE1/XBP1s/UBE2D3/NF-kB pathway is operating in these cells. How can this be explained? Only after XBP1s or UBE2D3 overexpression, NF-kB signaling appears to be affected.
-Fig 4: UBE2D3 and MIB1 The authors should discuss better what is the possible interaction between UBE2D3 and MIB1. As shown in fig4G, silencing of MIB1 cause a severe increase in UBE2D3 protein levels but this is not commented in the text.
-Fig 6: Chemokines driving recruitment of myeloid cells to UBE2D3 overexpressing tumors.
A formal demonstration that GL261-UBE2D3 tumors recruit higher numbers of MM and PNs through an enhanced production of CXCL2, IL-6 and/or IL-8 is lacking. For instance, they could compare the infiltration of myeloid cells in GL261-UBE2D3 vs GL261-UBE2D3-CXCL2KO tumors.Minor comments: important issues that can confidently be addressed.
Authors must provide replicates of the blots to sustain their claims: FigS1D, Fig3A, Fig3I, Fig4G. The authors should include a better description of the methods regarding bioinformatic analysis. For instance, which genes where used for MM/PN/T cell signatures in fig1A/S1A? Missing statistical significance on fig 2C and fig 6A to support their claims. Fig2F is presented in the text as mRNA levels but in the figure as protein levels.
Significance
Significance
In general, there is a clear interest both from academia and pharma companies to understand the role of the UPR in tumor biology and how the UPR shapes the immune compartment. This is highly relevant as the UPR is a novel drug target in cancer therapy, but unfortunately many inconsistent data are around. However, as the paper is now, it will not contribute to clarify these inconsistencies.
Compare to existing published knowledge.
Unfortunately, there are many studies around with inconclusive results and strong claims based on poorly validated tools.
Audience. Tumor immunologists, UPR field
Your expertise. Role of the UPR in immune cells and anti-tumor biology.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
Summary
In this manuscript, Obacz et al. investigated the role of IRE1 singaling in regulating the recruitment of myeloid cells in glioblastoma multiforme (GBM) microenvironment. They show that inhibition of IRE1 signaling decreased polynuclear neutrophil (PN) infiltration to GBM tumors in ananimal model; conversely, IRE1 activation correlated with higher expression of myeloid cells-attracting chemokines in GBM. They also show that IRE1-XBP1s pathway promotes proinflammatory chemokines in GBM tumor cells through upregulation of UBE2D3, which leads to degradation of the NFκB inhibitor IκB and activation of NFκB downstream signaling. …
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
In this manuscript, Obacz et al. investigated the role of IRE1 singaling in regulating the recruitment of myeloid cells in glioblastoma multiforme (GBM) microenvironment. They show that inhibition of IRE1 signaling decreased polynuclear neutrophil (PN) infiltration to GBM tumors in ananimal model; conversely, IRE1 activation correlated with higher expression of myeloid cells-attracting chemokines in GBM. They also show that IRE1-XBP1s pathway promotes proinflammatory chemokines in GBM tumor cells through upregulation of UBE2D3, which leads to degradation of the NFκB inhibitor IκB and activation of NFκB downstream signaling. Their finding of a novel IRE1/XBP1s/UBE2D3/NFκB axis is important for understanding the basis of pro-tumoral inflammation in GBM, potentially in other 'immune hot' cancers. The manuscript is well written and the conclusion is well supported by the experiments. However, there are a few critical points that need to be addressed to strengthen their study.
Major comments:
1.In this study, the authors used the GBM primary cell line RADH87 with stable overexpression of wild-type (WT) IRE1 or a truncated IRE1 variant. The expression of wild-type IRE1 was confirmed by Western analysis (Figure S1D). However, the expression of truncated IRE1 variant was not shown. In addition, without tunicamycin treatment, there was no visible difference in XBP1s expression between the cells expressing WT or the mutant IRE1. In the Boyden chamber assay (Figure 1C, D), conditioned medium from these cells were used; it was not described whether the cells were treated (e.g. with tunicamycin) to activate the IRE1 pathway.
2.The evidence that the mRNA expression of UBE2D3 positively correlates with IRE1/XBP1s pathway is weak. First, In Figure 3D, the correlation between the mRNA expression of UBE2D3 and XBP1 does not seem strong. In addition, as XBP1 mRNA level does not reflect IRE1 activation (as opposed to that of XBP1s), the level of XBP1s instead of total XBP1 should be assessed. Furthermore, such correlation should be validated in additional GBM cohorts/datasets.
3.The results in Figure 3 indicated that XBP1s acts as a transcriptional regulator of UBE2D3 expression. However, it is not clear whether this effect in GBM cells is direct or indirect. Further experiments such as chromatin immunoprecipitation and reporter assays are required to clarify this point.
4.In addition to UBE2D3, the two other ubiquitin-protein ligases, SYVN1 and UBE2J1, may also be implicated in the degradation of IκB. Did the authors assess their potential role on IκB degradation in their model system?
5.The authors only used ectopic expression of relevant proteins to test their hypothesis in U87 and RADH87 cells. It is necessary to validate these findings using siRNAs/inhibitors for IRE1 and UBE2D3 in a GBM cell line that expresses high levels of endogenous IRE1 and UBE2D3.
Minor comments:
1.In Figure 3I: The protein expression of UBE2D3 should be shown.
2.In the right panel of Figure 3I: What do the labels #1, 2, 5 mean? Clear descriptions should be provided in the figure legend.
3.In Figure S1D: The expression levels of the truncated IRE1 variant should be shown.
Significance
In this manuscript, the authors report some of the molecular mechanisms by which IRE1-XBP1s signaling controls GBM immune infiltration. They show that a novel IRE1/UBE2D3 signaling axis, mediated by XBP1s, regulates NF-κB activation, which subsequently promotes pro-inflammatory responses and the recruitment of immune/inflammatory cells to the tumor site. This study provides significant new information on the role of IRE1 in GBM. The findings also establish a basis for potential new approaches to improve the efficacy of current immunotherapies, also in other cancer types, which needs to be further explored.
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