Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

  1. Jinhai Deng
  2. Teng Pan
  3. Yourae Hong
  4. Zaoqu Liu
  5. Xingang Zhou
  6. Zhengwen An
  7. Lifeng Li
  8. Giovanna Alfano
  9. Gang Li
  10. Luigi Dolcetti
  11. Rachel Evans
  12. Jose M Vicencio
  13. Petra Vlckova
  14. Yue Chen
  15. James Monypenny
  16. Camila Araujo De Carvalho Gomes
  17. Kenrick Ng
  18. Caitlin McCarthy
  19. Xiaoping Yang
  20. Zedong Hu
  21. Joanna C. Porter
  22. Christopher J Tape
  23. Mingzhu Yin
  24. Manuel Rodriguez-Justo
  25. Sabine Tejpar
  26. Richard Beatson
  27. Tony Ng

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Abstract

Chemotherapy, the standard of care treatment for cancer patients with advanced disease, has been increasingly recognised to activate host immune responses to produce durable outcomes. Here, in colorectal adenocarcinoma (CRC) we identify chemotherapy-induced Thioredoxin Interacting Protein ( TXNIP), a MondoA-dependent tumor suppressor gene, as a negative regulator of Growth/Differentiation Factor 15 (GDF15). GDF15 is a negative prognostic factor in CRC and promotes the differentiation of regulatory T cells (Tregs), through CD48 ligation. Intriguingly, multiple models including patient-derived tumor organoids demonstrate that loss of TXNIP/GDF15 axis functionality is associated with advanced disease or chemotherapeutic resistance, with transcriptomic or proteomic GDF15/TXNIP ratios showing potential as a prognostic biomarker. These findings illustrate a potentially common pathway where chemotherapy-induced epithelial stress drives local immune remodelling for patient benefit, with disruption of this pathway seen in refractory or advanced cases.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    This well done and interesting paper examining the connection between TXNIP and GDF15. The main thrust is that TXNIP upregulation chemotherapies, such as Oxa, results in an a down regulation of GDF15 early in tumorigenesis. Later in tumorigenesis, TXNIP upregulation is less pronounced, elevating GFP15 resulting in a blockage of tumor suppressive immune responses. Generally the work is convincing. For example, it's clear that TXNIP is up regulated by Oxa in an ROS and MondoA-dependent manner. Likewise its quite clear TXNIP loss reads to an upregulation of GDF15. However, it's also quite clear that Oxa suppresses GDF15 in a manner that appears to be completely independent of TXNIP. The writing in the paper implies strongly that there is a mechanistic connection between TXNIP and GDF15, but no experiments investigate this possibility.

    We feel this is very fair and is reflective of a) perhaps an overemphasis of the TXNIP knockout observation and supportive tissue data, which suggests a relationship but not a mechanistic understanding b) an underemphasis of the data in Figure 3 that shows a decrease in GDF15 after oxaliplatin treatment in TXNIP knockout lines.

    We have addressed these concerns in several ways:

    1. We have carried out knockdown experiments using siRNA for ARRDC4, which we felt, given its regulation by MondoA and ROS, and homology to TXNIP, may also regulate GDF15. This was found to be the case and may explain the data in Figure 3. At the very least it shows that other factors involved in oxidative stress management may have similar impacts – a form of functional redundancy. Lines 553-559 “Finally, given our previous data (Figure S4) we looked to assess the role of ARRDC4 on GDF15 expression. In the absence of oxaliplatin, knocking down *ARRDC4 *in DLD1 and HCT15 cells drove an increase in GDF15. When challenged with oxaliplatin, both ARRDC4 and TXNIP expression increased and GDF15 decreased. When the ARRDC4 knockdown was challenged TXNIP increased further and GDF15 decreased further (Figure S6G-J). Given the common regulatory pathways and homology between TXNIP and ARRDC4, and their similar functional roles, we suggest these data are evidence of redundancy within this system. “

    We have included some context in the discussion:

    Lines 930-933: “Further support for both TXNIP and ARRDC4’s role in regulating GDF15 after the induction of ROS comes from a pan cancer meta-analysis assessing the impact of metformin (which has been reported to inhibit ROS) on gene expression. Here the top two downregulated genes were TXNIP and *ARRDC4 *and the top four upregulated genes were DDIT4, CHD2, ERN1 and GDF1572

    We have tempered the text:

    Lines 522-524 “It is important to note however that here we saw clear evidence that TXNIP was not solely responsible for the downregulation of GDF15 post oxaliplatin treatment, with decreased levels seen in knockout lines (Figure 3C-G, S5E).”

    Lines 926-929 “It must be stressed that these data do not place TXNIP as the sole regulator of GDF15, for example ARRDC4 can also be seen to regulate GDF15. We envisage TXNIP as one of a number of ROS-dependent GDF15 regulators, with this redundancy potential evidence of the importance of this regulatory framework.”

    We have carried out additional analysis detailed in the discussion and in Figure S12 which suggests TXNIP impacts MYC function, as reported elsewhere (detailed below). For ease, the key paper can be accessed through this link https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001778

    Lines 934-956: “The main shortcoming of this paper is the lack of mechanistic understanding linking TXNIP to GDF15. There are 650 transcription factors that have been shown, or are predicted, to bind to GDF15 promoter and/or enhancer regions. By assessing our list of differentially expressed genes (Suppl. Table 1-2) for the presence of these factors we identified 6 GDF15 binding TFs that show significantly decreased expression after oxaliplatin treatment in both cell lines (ATF4, MYC, SREBF1, PHB2, HBP1, KLF9). There was only one, MYC, that was downregulated by oxaliplatin treatment (validated; Figure S12A), and with this downregulation partially being rescued in a matched TXNIP knockout line (Figure S12B). We then observed that c-myc has been shown or is predicted to bind to promoter/enhancer regions of the top five transcriptomic and proteomic differentials in TXNIP knockout lines, including TXNIP itself (apart from C16orf90). Even with c-myc’s promiscuity (binds to 10-20% of all promoters/enhancers) this may be suggestive of a specific relationship. Finally, when looking at the correlations between these 6 TFs and TXNIP and GDF15 in the TCGA COAD dataset, MYC has the greatest and most significant negative correlation to TXNIP (r=-0.4631 p=1.42e-28) and the greatest and most significant positive correlation to GDF15 (r=0.4653 p=7.32e-29). ATF4 and PHB2 are the other TFs in the list, that show the same significant trends (Figure S12C), and therefore may play a role in the TXNIP-independent oxaliplatin-dependent regulation of GDF15. Further exploration of these additional TFs is outside the scope of the current manuscript.

    MYC’s role in bridging from TXNIP to GDF15 is further supported by a recent paper which shows that TXNIP is “a broad repressor of MYC genomic binding” and that “TXNIP loss mimics MYC overexpression”73. Furthermore, the inter-dependent regulatory relationship between MondoA, TXNIP, and MYC has been seen in a variety of models74, whilst the impact of NAC on MYC-dependent pathways has been seen in lymphoma75. These studies lend credence to the idea that MYC is the most likely TXNIP-regulated TF that regulates GDF15 in our systems.”

    It seems equally likely that TXNIP and GDF15 represent independent parallel pathways. Even if TXNIP is a direct regulator of GDF15, it's also clear that other "factors" up or down-regulated by Oxa also contribute to the regulation of GDF15. These are not explored and even though TXNIP is highly regulated genes shown Figure 2 that are not identified or discussed that may also be contributing to GDF15 regulation.

    As mentioned above, the new data suggests that at least one other factor, ARRDC4, can regulate GDF15 (changes upon oxaliplatin treatment) and that MYC is a potential mechanistic bridge between TXNIP and GDF15. Whilst assessing for the transcription factor that may link TXNIP and GDF15 we found an additional 5 TXNIP-independent factors (ATF4, PHB2, SREBF1, HBP1, KLF9) that bind to GDF15 promoter/enhancer regions and are downregulated post-oxaliplatin treatment. When looking at correlations between these factors and GDF15 in the TCGA COAD dataset, ATF4 and PHB2 correlate most closely with GDF15 (when removing MYC) and so we would cautiously suggest that these may be the most pertinent. This data is now included.

    Further, the experiments treating PBMCs with conditioned media contain other cytokines/factors, in addition to GDF15, that likely also contribute the observed effects on the different immune cells understudy. The conditioned media from GDF15 knock out cells are a good experiment, but the media is not rigorously tested to see what other cytokines/factors might have also been depleted.

    The TXNIP knockout media is the same as that analysed by mass spec and the protein array, however as the reviewer states there is no analysis (excluding assessing for the presence or absence of GDF15) on the double knockout supernatant or over-expression supernatant. The text has been corrected as follows:

    Lines 675-679. “In light of other secreted factors being seen to be regulated by TXNIP (Figure 3A-B), we included double knockouts (TXNIP and GDF15 knockout; GTKO) as well as an overexpression system (GDF15a) to test for GDF15 specific effects. However, we do not know the impact of knocking out or overexpressing GDF15 on the broader secretome.”

    Perhaps a GDF15 complementation experiment would help here.

    We felt that the association between GDF15 and Treg induction is reasonably well established in the literature, and so once we saw that the supernatant from our GDF15 overexpression system (+/- CD48 blockade) complemented what has already been demonstrated, we were encouraged. However we needed more – hence the TCGA data and IHC staining.

    Finally, even if completely independent, a TXNIP/GDF15 ratio does seem to have utility in determining chemo-therapeutic response.

    We agree – we feel that conceptually this may be the most interesting part of the project and is an example of what can be done with these tools.

    Other major points:

    1. Please label the other highly regulated genes shown in Fig 2A and B. Might they also explain some of the underlying biology. This could be on the current figures or in a supplement, though the former is preferred.

    Many thanks – we have done this.

    Please address why the TXNIP induction is so much less in patient-derived organoids vs. cell line spheroids (Fig S2). By the western blots, TXNIP inductions in the organoids looks quite modest. Further, the text is quite cryptic and implies that the "upregulation" is similar in both organoids and spheroids.

    You are absolutely correct. Many apologies, the wording has changed:

    Lines 320-323 “In both models we observed the upregulation of TXNIP mRNA (Figure S2E-H) and TXNIP protein (Figure S2I-L) after oxaliplatin treatment, with spheroids showing greater responsiveness. This difference is most likely due to culturing conditions or differences in the number and location of cycling cells.”

    We have two possible explanations. Firstly the media in which the organoids are cultured contains a lower glucose concentration than that used for the spheroids. As per some of our new data (Figure S3 – later in the rebuttal), the upregulation of TXNIP after oxaliplatin is glucose dependant, with lower concentrations resulting in less of a differential. Secondly, while restricted to the periphery, the Ki67 signal in DLD1 spheroids is quite pronounced indicating that, within the outer zone, many cells (probably the majority) are in the S/G1/G2 phase of the cell cycle at any given point in time (figure below this text).

    This is not the case for the organoids, where the Ki67 (and pCDK1) signal is quite weak, and only sporadic in the outer layer. So we believe that there are many more rapidly cycling cells in the most drug-exposed layer of spheroids when compared to the comparable region in organoids. As the spheroid cells are likely cycling more rapidly, they would also be expected to be more adversely affected by the drug within the finite drug treatment window. Indeed, these spheroids grow large, and quite quickly. If the organoid cells are cycling more slowly and if, within the cell layer most exposed to drug, these cycling cells are less abundant, then the TXNIP response may well be subdued in organoids when compared with spheroids.

    We have decided to not include the above (full) explanation and figure within the new draft, as we feel it may distract from the central message. However do let ourselves and the editor know if you disagree.

    What was the rationale of performing the MS experiment on control and TXNIP KO DLD1 cells in the absence of oxaliplatin? The other experiments in Fig 3 clearly show that Oxa can repress GDF15 even in the absence of TXNIP, which implicates other pathways. ARRDC4? Or something else? This needs to be addressed.

    We adopted this approach because of the order in which the assays occurred and technical issues surrounding the use of post-oxaliplatin treated supernatant. By the time we moved to the proteomics we had already identified, and validated, GDF15 as our number one candidate (initially from the protein array), in terms of response to oxaliplatin and dependence on TXNIP. This led us to the next stage of the project – to assess the environmental impacts of this factor in vitro before validation in situ. To do this, aware of the issue of contaminated recombinant GDF15, we decided early on to use cell line supernatant. We carried out some pilot studies on immune cells using supernatant from oxaliplatin treated cell lines and we had several technical issues (difficulty in determining the correct controls, immune cell death…). This changed the emphasis to using supernatant from knockout models rather than knockout and treated models. Before we began these assays in earnest we wanted to assess exactly what was enriched in TXNIP knockout supernatant and so we turned to proteomics. When this further validated GDF15, we then generated GDF15 and TXNIP/GDF15 knockouts to further elucidate GDF15’s role specifically.

    With regards the other pathways, as you correctly predicted, ARRDC4 also appears to regulate GDF15 – many thanks for helping with this line of enquiry. Please see earlier in the rebuttal for more details and the data.

    The data in 3J with the MondoA knockdown is not convincing. The knockdown is weak and TXNIP goes down a smidge. Agree that GDF15 goes up

    We agree. We have re-run this and pooled the densitometry data – see new figure below (Panel 3J).

    Minor points

    1. Line 79. The "loss" of TXNIP/GDF15 axis is confusing. It's really loss of TXNIP and upregulation of GDF15, right?

    Absolutely - corrected to responsiveness.

    Lines 144-147: “Intriguingly, multiple models including patient-derived tumor organoids demonstrate that the loss of TXNIP and GDF15 responsiveness to oxaliplatin is associated with advanced disease or chemotherapeutic resistance, with transcriptomic or proteomic GDF15/TXNIP ratios showing potential as a prognostic biomarker.”

    Please provide an explanation for the different stages in tables 1 and 2. This will likely not be clear to non-clinicians.

    Many thanks. The following has been added at the bottom of the second table.

    Lines 304-309: “The TNM staging system stands for Tumor, Node, Metastasis. T describes the size of the primary tumor (T1-2; 5cm). N describes the presence of tumor cells in the lymph nodes (N0; no lymph nodes. N1-3 >0). M describes whether there are any observable metastases (M0; no metastases. M1; metastases). The clinical stage system is as follows: I/II; the tumor has remained stable or grown, but hasn’t spread. III/IV; the tumor has spread, either locally (III) or systemically (IV).”

    Line 231 should probably read ...cysteine (NAC), a reactive oxygen species inhibitor,

    Many thanks - corrected

    Line 247, should be RT-qPCR I think.

    Many thanks - corrected

    Lines 343-345. I don't quite understand the wording. Does this mean to say that 675 soluble proteins were not changed between the condition media from both cell populations?

    Yes, exactly this. We have removed as this is superfluous and confusing.

    The data in FigS1 B and C don't seem to reach the standard p value of > 0.05

    Very true – we have rewritten the text to make sure the reader knows there is no significance.

    Lines 269-271. “High levels of both the protein (significantly) and the transcript (not significantly) were seen to be associated with favourable prognosis (Figure 1G,H and S1B,C).”

    **Referee Cross-Commenting**

    cross comment regarding referees 2 and 3 above. I'm am convinced that TXNIP is at least contemporaneously upregulated with GDF15 downregulation. However, the strong implication from the writing is that TXNIP regulates GDF15 directly. I agree with the comment above that exploring mechanisms may be open-ended especially as TXNIP has been implicated in gene regulation by several different mechanism. I'd be satisfied with a more open-minded discussion of potential mechanisms by which TXNIP may repress GDF15 and the possibility of other parallel pathways that likely contribute to GDF15 repression.

    Many thanks, this is a generous and understanding approach. As described above we have carried out extra analysis and have found 6 differentially regulated transcription factors which have been shown to bind GDF15 promoter or enhancer regions with 1 of these, MYC, being significantly affected in the TXNIP knockout cell lines, which in combination with supportive literature suggests a degree of TXNIP dependence. We have also identified ARRDC4 as an additional regulator of GDF15 – again please see above.

    Reviewer #1 (Significance (Required)):

    This is an interesting contribution but the mechanistic connection between GDF15 and TXNIP is relatively weak. That said, even as independent variables they do seem to have utility in predicting therapeutic response.

    Many thanks for the comment – we concur. We have reanalysed our data looking for relevant transcription factors (those that bind GDF15 promoter / enhancer regions) finding MYC as the most likely bridge. Please see above.

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

    The manuscript by Deng et al. investigates a mechanistic link between TXNIP and GDF15 expression and oxaliplatin treatment and acquired resistance. They observe an upregulation in TXNIP expression in the tumors of patients who have previously received chemotherapy. They demonstrate oxaliplatin-driven MondoA transcriptional activity is what underlies the induction of TXNIP. They further demonstrate that TXNIP is a negative regulator of GDF15 expression. Together, oxaliplatin induces MondoA activity and TXNIP expression, resulting in a downregulation of GDF15 expression and consequently decreased Treg differentiation.

    Major Comments

    1. The authors suggest that TXNIP induction and GDF15 downregulation are a common effect of chemotherapies; however, the mechanistic studies were limited to oxaliplatin. The authors should clarify this point through further investigation using other commonly used CRC chemotherapies (5-FU, irinotecan, etc.),or through textual changes. To be clear, I think that the oxaliplatin results could potentially stand on their own but would require additional clarification. For example, regarding the patient samples analyzed in 1D and 4F, which patients received oxaliplatin? Could the analysis of publicly available molecular data be drilled down to just the patients who received oxaliplatin?

    Many thanks – this is an excellent point. Firstly, all the patients in 1D and 4F received oxaliplatin. Secondly, we have included new data looking at the impact of other chemotherapies (FOLRIRI, FU-5 and SN-38) on aspects of the study, ultimately finding that these processes (especially an anti-correlation between GDF15 and TXNIP changes upon chemo treatment) appear to be specific to oxaliplatin. These data have been added (Figure S11) and throughout the emphasis has been switched from chemotherapeutic treatment to oxaliplatin treatment.

    Lines 796-799: “To check if the pre-treatment GDF15/TXNIP ratio could be used for patients treated with FOLFIRI we performed the same analyses finding no significance (S11A-D). This oxaliplatin specificity was then confirmed by western blot analysis in DLD1 and HCT15 cells treated with 5-FU or SN38 (Figure S11E-F).

    Example of change of emphasis from ‘chemotherapy’ to ‘oxaliplatin’ – lines 139-142: “Here, in colorectal adenocarcinoma (CRC) we identify oxaliplatin-induced Thioredoxin Interacting Protein (*TXNIP), *a MondoA-dependent tumor suppressor gene, as a negative regulator of Growth/Differentiation Factor 15 (GDF15).”

    The data demonstrating the induction of MondoA transcriptional activity and TXNIP expression in response to oxaliplatin treatment is quite convincing. The data regarding ROS induction of TXNIP is interesting, especially in light of other studies arguing that ROS limits MondoA activity (PMID: 25332233). Given this apparent disparity, I think that this study could really be strengthened by also investigating other potential mechanisms of oxaliplatin induction of MondoA. In particular, given many studies arguing for direct nutrient-regulation of MondoA, the authors should address the potential for oxaliplatin regulation of glucose availability and a potential glucose dependence of oxaliplatin-induced TXNIP. 2

    In line with the previous point, since MondoA activity and TXNIP expression are sensitive to glucose levels, the authors should investigate oxaliplatin-regulation of TXNIP under physiological glucose levels. No need to replicate everything, just key experiments.

    We feel these are excellent point and really help the piece – many thanks. We have carried out assays around these points suggested and have included the findings in the new draft – see below.

    Lines 332-339: “As such, we went back to first principles and assessed the impact of different concentrations of glucose on TXNIP induction +/- oxaliplatin treatment, finding a concentration dependent effect (Figure S3A). Intriguingly, high glucose alone was able to induce increased TXNIP expression. We then assessed if oxaliplatin treatment drove an increase in glucose uptake, with this seen at concentrations >10mM (Figure S3B). Next, to investigate the impact of glucose metabolism, and consequent ROS generation, on TXNIP induction we treated cells with Antimycin A, an inhibitor of oxidative phosphorylation, finding a complete block in oxaliplatin-induced TXNIP (Figure S3C).”

    The authors did a good job of linking TXNIP and GDF15 in untreated conditions; however, the data arguing for oxaliplatin regulation of GDF15 through TXNIP is less clear. For example, in 3B-H, oxaliplatin treatment reduces GDF15 approximately to the same extent in the NTC and TKO cells, potentially in line with a mechanism of downregulation that doesn't involve TXNIP.

    A very salient point and completely in line with the other reviewers. We have carried out a few additional analyses mentioned previously in this letter. The most pertinent for this specific point are the experiments around ARRDC4, where we found evidence to suggest that, like TXNIP, it regulates GDF15.

    Minor Comments

    1. The presentation of data in Figure 5 is confusing. A-B include raw cell numbers, whereas C-F show "normalized proliferation." What does this mean? And how was the normalization done?

    Apologies for this. Legend test has been corrected to “Normalised proliferation (normalised to MFI from control: i.e. cells treated with supernatant from NTC cells) on gated CD3+CD8+ or CD3+CD4+ cells is shown. n=6. (G-H) Normalised IFNg concentrations (normalised to MFI from control: i.e. cells treated with supernatant from NTC cells) in the supernatant of cells from C-F.” (lines 727-729).

    **Referee Cross-Commenting**

    cross-comment regarding reviewer #1

    I agree with the referee that the link between TXNIP and GDF15 is weak, though as I mentioned before, this is particularly true in the context of oxaliplatin-regulation of TXNIP. I agree that given all the presented data, it is likely that oxaliplatin-regulation of TXNIP and GDF15 are independent. In my opinion, the referee brought up all valid concerns, but this is by far the biggest concern that I share.

    We agree that this is the weakest aspect of the paper, however our new analyses plus supportive literature, suggests that the relationship between TXNIP and GDF15 may be mediated by MYC (please see above)

    cross-comment regarding reviewer #3

    The major concern that this referee addresses is whether another transcription factor supersedes the proposed MondoA/TXNIP induction in regulating GDF15 expression in later stage CRC. In my opinion, this another other concerns of the referee are all valid, but still I remain unconvinced that TXNIP induction underlies the oxaliplatin-regulation of GDF15. I think fleshing out that aspect of the study would potentially help the authors tease apart how this potential MondoA-TXNIP-GDF15 axis is dysregulated later in CRC progression.

    This is a great discussion. Interestingly enough, c-myc is seen at higher levels in late stage CRC (Hu X, Fatima S, Chen M, Huang T, Chen YW, Gong R, Wong HLX, Yu R, Song L, Kwan HY, Bian Z. Dihydroartemisinin is potential therapeutics for treating late-stage CRC by targeting the elevated c-Myc level. Cell Death Dis. 2021 Nov 5;12(11):1053. Doi: 10.1038/s41419-021-04247-w. PMID: 34741022; PMCID: PMC8571272.), is seen as an important factor in resistance, and as this review argues, is driven by stress (Saeed H, Leibowitz BJ, Zhang L, Yu J. Targeting Myc-driven stress addiction in colorectal cancer. Drug Resist Updat. 2023 Jul;69:100963. Doi: 10.1016/j.drup.2023.100963. Epub 2023 Apr 20. PMID: 37119690; PMCID: PMC10330748.). So it is very plausible that the partial TXNIP-mediated regulation of myc in early / sensitive CRCs that we may be observing, and has been reported recently (TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding Lim TY, Wilde BR, Thomas ML, Murphy KE, Vahrenkamp JM, et al. (2023) TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding. PLOS Biology 21(3): e3001778. https://doi.org/10.1371/journal.pbio.3001778) is lost in late stage / resistant CRCs. If this is the case, in effect what we would have observed is the loss of a stress-associated method (TXNIP) of controlling c-myc activity. What makes our collective lives difficult is that, as reported “this expansion of Myc-dependent transcription following TXNIP loss occurs without an apparent increase in Myc’s intrinsic capacity to activate transcription and without increasing Myc levels.” (TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding Lim TY, Wilde BR, Thomas ML, Murphy KE, Vahrenkamp JM, et al. (2023) TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding. PLOS Biology 21(3): e3001778. https://doi.org/10.1371/journal.pbio.3001778)

    Reviewer #2 (Significance (Required)):

    Generally speaking the experiments are well controlled and the findings are significant and novel. Though the link between MondoA activity and ROS could be strengthened, and the data could be validated under more physiological settings. Further, the authors should clarify their interpretations so as to not overstate the findings.

    Many thanks for the comments. We have taken onboard the need for more physiological settings and have included varying levels of glucose to reflect concentrations in different environments. We have repeated the siMondoA work in 3J strengthening the conclusions wrt its impact on TXNIP and GDF15 expression (see above).

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this well-written manuscript, the authors show that chemotherapy increases a MondoA-dependent oxidative stress-associated protein, TXNIP, in chemotherapy-responsive colorectal cancer cells. They show that TXNIP negatively regulates GDF-15 expression. GDF-15, in turn, correlates with the presence of T cells (Treg), and inhibits CD4 and CD8 T cell stimulation. In advanced disease and chemo-resistant cancers, upregulation of TXNIP and downregulation of GDF-15 appear to get lost. Based on a somewhat smallish data set, the authors suggest that the pre-treatment GDF-15/TXNIP ratio can predict responses to oxaliplatin treatment. This is a very interesting, novel finding. In general, the quality of the experiments and the data are high and the conclusions appear sound. Still, there are a number of aspects that should still be improved:

    The observed loss of the ROS - MondoA - TXNIP - GDF15 axis in chemoresistant and/or metastatic tumors implies that another transcription factor or pathway becomes dominant upon tumor progression. As this switch would be key to better understanding the mechanism underlying the prognostic role of the TXNIP/GDF15 ratio, the authors should at least do data mining followed by ChEA or Encode (or other) analysis to identify transcription factors or pathways that become activated in late-stage/metastatic CRC cells. There is a high likelihood that a transcription factor or pathway involved in GDF-15 upregulation in cancer (e.g. p53, HIF1alpha, Nrf2, NF-kB, MITF, C/EBPß, BRAF, PI3K/AKT, MAPK p38, EGR1) supersedes the inhibitory effect of the MondoA-TXNIP axis. As it stands, the proposed loss of function of the ROS - MondoA - TXNIP - GDF-15 axis is far less convincing than almost all other aspects of the study.

    An extremely fair point. We adopted a similar approach to that suggested – as mentioned above, we looked at TFs that bind to GDF15 promoter/enhancer regions and then looked at the presence of these in our transcriptomic data – specifically any evidence of change post oxaliplatin treatment. We found 6 such TFs that were decreased post-oxaliplatin treatment. We then looked for any evidence of TXNIP dependence in these TFs by comparing post-oxaliplatin treatment across NTC and TXNIP knockout lines, when we did this we found only one GDF15 promoter/enhancer binding TF was significantly changed: MYC. We then looked at the relationship between MYC,TXNIP, and GDF15 against the other 5 ‘control’ TFs in the TCGA COAD dataset, we found that MYC showed the strongest correlations, in the ‘correct’ directions. This finding was further backed up in the literature where a TXNIP knockout in a breast cancer model drove c-myc-dependent transcription, whilst c-myc has been observed to increase in later stage CRC patients, is associated with cellular stress and resistance. The collective evidence therefore suggests that MYC is the factor that is initially at least partially regulated by TXNIP, before this regulation is lost in advanced / resistant disease. Continuing on this line, it is likely that the predictive GDF15/TXNIP ratio is at least in part, a measure of c-myc responsiveness to oxaliplatin. All the while we must bear in mind TXNIP-independent oxaliplatin-dependent regulation of GDF15, most likely ARRDC4, as described earlier in this document.

    Using pathway analysis software to compare our transcriptomic data from cell lines treated with/without oxaliplatin, the most likely pathways upstream of MYC/c-myc that are negatively affected by chemotherapy are BAG2, Endothelin-1, telomerase, ErbB2-ErbB3 and Wnt/B-catenin. When looking at the comparison of UTC and resistant lines’ transcripts there is only one key component of these pathways which is upregulated in both lines - ERBB3 – which has already been shown to be important in CRC metastasis and resistance (Desai O, Wang R. HER3- A key survival pathway and an emerging therapeutic target in metastatic colorectal cancer and pancreatic ductal adenocarcinoma. Oncotarget. 2023 May 10;14:439-443. doi: 10.18632/oncotarget.28421. PMID: 37163206; PMCID: PMC10171365.). It is highly speculative, but our data suggests the most likely pathway to supersede TXNIP in its (partial) regulation of MYC is the ErbB2-ErbB3 pathway.

    My further criticisms are mostly more technical:

    Figure 2 I-L: What was the extent of MondoA downregulation achieved by siRNA treatment? Could the effects also be seen with the small molecule mondoA inhibitor SBI-477 (or a related substance)?

    This experiment has been repeated. The pooled densiometric data is also now given (please see above).

    How do you explain the different GDF-15 levels between untreated non-target control cells (NTC) and TXNIP knock-down cells (TKO) in Figures 3C-F?

    The only way to interpret this is that there is a TXNIP-independent pathway regulating GDF15 expression after oxaliplatin treatment, as described this is most likely to be ARRDC4 - the text has been updated to:

    Lines 522-524: “It is important to note, however, that we saw clear evidence that TXNIP was not solely responsible for the downregulation of GDF15 post oxaliplatin treatment (Figure 3C-G, S6E).”

    In figures 3 E-G the dots for the individual measurements should be indicated. This would be more informative than just the bar graphs.

    Completed.

    Figure 4C,D and Table 3: Data on the role of GDF-15 in CRC are largely valedictory of previous work (e.g. Brown et al. Clin Cancer Res 2003, 9(7):2642-2650, Wallin et al., Br J Cancer. 2011 May, 10;104(10):1619-27). Therefore, the previous studies should be cited.

    Apologies for the oversight and many thanks – this is an excellent addition.

    Figure 5C-F: Please indicate in the figure legend how proliferation was assessed.

    Many thanks. This was noticed by another reviewer also. We have changed the text to include how the data was normalised: “(C-F) Labelled PBMCs were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of fresh supernatant from indicated cell lines, before being stained with anti-CD3 and anti-CD8 (C-D) or anti-CD4 (E-F) antibodies and measured by flow cytometry. Normalised proliferation (normalised to MFI from control: i.e. cells treated with supernatant from NTC cells) on gated CD3+CD8+ or CD3+CD4+ cells is shown. n=6. (G-H) Normalised IFNg concentrations (normalised to MFI from control: i.e. cells treated with supernatant from NTC cells) in the supernatant of cells from C-F.” (lines 724-730)

    Figure S8E-G: Please indicate the analysed parameters in the graphs. In Figure S8G, the legend just indicates that "aggression of tumour" is dichotomized and plotted. This clearly requires a better definition.

    Many thanks, this has been changed as per the below.

    Lines 862-868: “(E-G) Receiver operating characteristic (ROC) curves showing area under the curve and p values for the use of GDF15/TXNIP ratio in predicting origin of cell line (E; primary; DLD1, HCT15, HT29, SW48 [n=4] or secondary; DiFi, LIM1215 [n=2]), sensitivity to oxaliplatin (F; parental DLD1 (plus biological repeat), HCT15 [n=3] or resistant DLD1 (plus biological repeat), HCT15 [n=3]), aggression of tumor (G; non-aggressive; The authors propose a novel ROS - MondoA - TXNIP - GDF15 - Treg axis, where MondoA activation, TXNIP up- and GDF-15 downregulation enhance tumor immunogenicity. While this axis has been analyzed in some detail, GDF-15 is not only linked to induction of regulatory T cells. There has been a report showing that GDF-15/MIC-1 expression in colorectal cancer correlates with the absence of immune cell infiltration (Brown et al. Clin Cancer Res 2003, 9(7):2642-2650). The link between GDF-15 and immune cell exclusion has also been confirmed in other conditions, including different cancers (Kempf et al. Nat Med 2011, 17(5):581-588, Roth P et al. Clin Cancer Res 2010, 16(15):3851-3859, Haake et al. Nat Commun 2023, 14(1):4253). A key mechanism is the GDF-15 mediated inhibition of LFA-1 activation on immune cells. As the authors argue that the described pathways turns cold tumors hot in response to oxaliplatin-based chemotherapy, this GDF-15 dependent immune cell exclusion mechanism might be at least as relevant than induction of Treg. Likewise, inhibition of dendritic cell maturation by GDF-15 (Zhou et al. PLoS One 2013, 8(11):e78618) could explain why GDF-15high tumors are immunologically cold. Reviewed in 3

    The authors propose that the pathways discovered by them contributed to the "heating up" of the tumor microenvironment after oxaliplatin-based chemotherapy. The authors should thus look in their data sets for the presence of cytotoxic T cells and their possible correlation with TXNIP and GDF-15 levels.

    This is a wonderful explanation – many thanks. We have taken the opportunity to assess the impact of GDF15 expression on a variety of T cell markers (Figure S9). In this data a negative association between GDF15 and CD8 CTLs can clearly be seen, as predicted by the reviewer.

    Lines 712-717: “To assess if the GDF15-dependent presence of Tregs may be associated with a decrease in activated cytotoxic CD8 T cells, we interrogated the TCGA COAD dataset. We found that low GDF15 tumors carried significantly higher levels of CD8, CD69, IL2RA, CD28, PRF1, GZMA, GZMK, TBX21, EOMES and *IRF4 *(Figure S9); transcripts indicative of activated cytotoxic CD8 T cells. High GDF15 tumors were enrichment for FOXP3 and, interestingly, *RORC *(Figure S9). These data support the hypothesis that GDF15 induces Foxp3+ve Tregs which inhibit CD8 T cell proliferation and activation in the TME.”

    The paragraph on GDF-15 receptors needs to be corrected: The purported role of a type 2 transforming growth factor (TGF)-beta receptor in GDF-15 signalling had been due to a frequent contamination of recombinant GDF-15 with TGF-beta (Olsen et al. PLoS One 2017, 12(11):e0187349). There have been a number of screenings for GDF-15 receptors that have all failed to show an interaction between GDF-15 and TGF-beta receptors. Instead, only GFRAL was found in these large-scale screenings (Emmerson et al. Nat Med 2017, 23(10):1215-1219, Hsu et al. Nature 2017, 550(7675):255-259, Mullican et al. Nat Med 2017, 23(10):1150-1157, Yang et al. Nat Med 2017, 23(10):1158-1166). The one subsequent report that shows a link between GDF-15, engagement of CD48 on T cells and induction of a regulatory phenotype (Wang et al. J Immunother Cancer 2021, 9(9)) still awaits independent validation. Considering that CD48 lacks an intracellular signaling domain that would be critical for a classical receptor function, I recommend to be more cautious regarding the role of CD48 as GDF-15 receptor. Given the mechanism outlined by Wang et al. the word interaction partner might be more apt. Moreover, an anti-GDF-15 antibody would be a good control for the experiments involving an anti-CD48 antibody in Figure 5.

    Thank you so much for this concise and highly informative paragraph. We have changed the text to read:

    202-204: “As a soluble protein, GDF15 exerts its effects by binding to its cognate receptor, GDNF-family receptor a-like (GFRAL)44,45,46,47 or interaction partner, CD48 receptor (SLAMF2)43, with the latter still requiring additional verification.”

    We would have ideally included an anti-GDF15 antibody in the CD48 assay at the time but didn’t have the foresight. We have included the additional text to temper any conclusions.

    Lines 701-711: “Furthermore, when stimulating naïve CD4 T cells in the presence of GDF15 enriched supernatant we were able to both differentiate these cells into functional Tregs and also block the generation of this functionality using an anti-CD48 antibody (Figure 5M-N). However, it must be stressed that the binding and functional impacts of GDF15’s interaction with CD48 still require further verification.”

    Cell surface externalization of annexin A1 has been described as a failsafe mechanism to prevent inflammatory responses during secondary necrosis (PMID: 20007579). Thus, I am surprised that the authors list annexin A1 among the immune-stimulatory molecules exposed or released in response to chemotherapy-induced cell death (line 103). Please clarify!

    We agree – it shouldn’t be there!! Removed. Many thanks.

    **Referee Cross-Commenting**

    Regarding the cross-comment by referee 2: In my opinion, the data shown in Figure 3C-H clearly demonstrates that TXNIP can repress GDF-15 expression. I agree that there will likely be further regulators. The GDF-15 promoter is constantly regulated by a multitude of factors (which mostly induce transcription). As downregulation of GDF-15 in response to oxaliplatin is the opposite of the frequently described induction of GDF-15 upon chemotherapy, net effects may always be "smudged" by contributions from different pathways (e.g. by cell stress due to siRNA transfection). Therefore, I believe that the data are as good as it will get. Accordingly, I would not force the authors to further amplify the observed effect.

    Many thanks for your understanding – yes, GDF15 has >650 TFs that bind its promoter/enhancer regions – a number we found rather daunting. Happily your comments and those of the other reviewers inspired us to dig and we now have data that is supportive of MYC’s and ARRDC4’s involvement – detailed throughout this reply.

    cross comment regarding referee #1: I share the general assessment of the referee and recognize the very detailed mechanistic analysis. To further support the moderate effects of the MondoA knockdown, a small molecule inhibitor like SBI-477 might be useful. (I had already suggested using this inhibitor to support these data.)

    Many thanks for the suggestion. We opted to increase the number of siRNA repeats instead – with the data included in Figure 3J (above).

    Still, my view on the potential relevance of oxaliplatin-induced, TXNIP-independent downregulation of GDF-15 differs from that of referee 1. In the clinics, platinum-based chemotherapy is one of the strongest inducers of GDF-15 (compare Breen et al. GDF-15 Neutralization Alleviates Platinum-Based Chemotherapy-Induced Emesis, Anorexia, and Weight Loss in Mice and Nonhuman Primates. Cell Metabolism 32(6), P938-950, 2020.DOI:https://doi.org/10.1016/j.cmet.2020.10.023). I was thus surprised that the authors found a pathway, which leads to an outcome that an exactly opposite effect.

    This is fascinating that oxaliplatin drives this increase in GDF15 – we were unaware of this paper. Looking at figure 2(H-K), GDF15 is being produced from multiple non-diseased tissues after systemic chemotherapy – even at day 19 post-treatment – this suggests that wrt this study, systemic GDF15 could not be used as a readout of success or otherwise – which is extremely helpful! Thank you.

    Thus far, the only obvious reason for reduced GDF-15 secretion upon treatment with cytotoxic drugs was a reduction in tumor cell number due to cytotoxicity.

    Please do not discount this. This study was focused on the cells which survived oxaliplatin treatment – the cells which did not were discarded. Our view, given your input, would be a complex picture where in early stages systemic GDF15 goes up, due to off-target effects, but locally levels drop owing to cell death and this, and other, stress-related pathways in the remaining tumor cells.

    Still, the authors managed to convince me that the described pathway (ROS - MondoA - TXNIP - GDF-15) exists. (Here, I still largely concur with referee 1.) Moreover, as we have identified some factors required for GDF-15 biosynthesis that could easily interact with TXNIP, I find the proposed mechanism plausible.

    Extremely encouraging for us to hear!

    Nevertheless, as a downregulation of GDF-15 in response to chemotherapy is hardly ever observed in late-stage cancers, I believe that the observed switch in pathway activation between early- and late-stage cancers might be highly relevant - in particular, as there is so much evidence for platinum-based induction of GDF-15 in late-stage cancer patients. Emphasizing the divergent clinical observations (e.g. by Breen et al.) could thus help to put the finding into perspective.

    Very much agree. We did see this phenomenon in LIM1215 cells (Figure 6B) and the resistant lines we generated continually produced higher levels.

    Analysing TXNIP-independent mechanisms involved in the oxaliplatin-dependent repression of GDF-15, as suggested by referee #1, will require enormous efforts and resources, and may still turn out to be fruitless. Personally, I would thus be content if the authors just mentioned possible contributions from other pathways upon cancer progression. To me, the described pathway seems to be limited to early-stage cancers, and the actual finding that GDF-15 is downregulated is an interesting observation, irrespective of further involved pathways.

    Many thanks – this is extremely fair. Happily we have managed to make some tentative steps forward in highlighting the potential role of MYC, and the suggestion of redundancy wrt ARRDC4, but as you say, much more work needs to be done to fully understand these processes.

    cross comment regarding referee #2: I fully agree with the referee that activation of the pathway by further chemotherapeutic drugs could be a valuable addition. As Guido Kroemer´s lab has described oxaliplatin to induce a more immunogenic cell death compared to other platinum-based chemotherapies, even a rather limited comparison between oxaliplatin and cisplatin could be very interesting.

    Absolutely agree – extra data on this has been included in Figure S11, which is included earlier in this letter. We also uncovered a meta-analysis using metformin, which has been seen to inhibit ROS, where TXNIP and ARRDC4 are the top two downregulated transcripts whilst GDF15 appears in the top four upregulated. This may suggest that chemotherapeutic immunogenicity, at least through the presence or absence of GDF15, may in part be driven by ROS.

    Lines 930-933: “Further support for both TXNIP and ARRDC4’s role in regulating GDF15 after the induction of ROS comes from a pan cancer meta-analysis assessing the impact of metformin (which has been reported to inhibit ROS) on gene expression. Here the top two downregulated genes were TXNIP and *ARRDC4 *and the top four upregulated genes were DDIT4, CHD2, ERN1 and GDF1572 “

    Reviewer #3 (Significance (Required)):

    In general, this is a very interesting manuscript describing a cascade of events that may contribute to successful chemotherapy (which likely requires induction of an immune response against dying tumor cells.) The observation that this pathway is only active in early/non-metastatic cancer cells is striking. Unfortunately, the authors cannot explain inactivation of this pathway in later stage/ metastatic/ highly aggressive cancers. Understanding this switch could easily be the most important finding triggered by this report. Therefore, I highly recommend to make some effort in this direction. Strikingly, the authors find that disruption of TXNIP-mediated GDF-15 downregulation is strongly associated with worse prognosis. They also suggest that this ratio could indicate whether a patient will respond to oxaliplatin-based chemotherapy.

    This is again very fair – we have posited a potential mechanism for the loss of this switch elsewhere in this reply– one which involves a change in TXNIP-mediated MYC regulation and/or increased HER2-HER3 signalling – but although reasonable for a rebuttal (and publication in that context) we do not feel we have the evidence to include this within the full manuscript.

    Altogether, the findings described in manuscript are very novel and may have prognostic (or, in case of the presumed loss of the MondoA - TXNIP - GDF-15 pathway) therapeutic implications. Thus, the manuscript certainly fills various gaps and should be of major interest for cell biologists working on immunogenic cell death, or colorectal cancer, or MondoA, TXNIP or GDF-15. Still, due to its translational implications, it would also be worthwhile reading for a large number of researchers in the oncology field.

    We are very grateful for your kind comments.

    1 Sinclair, L. V., Barthelemy, C. & Cantrell, D. A. Single Cell Glucose Uptake Assays: A Cautionary Tale. Immunometabolism 2, e200029, doi:10.20900/immunometab20200029 (2020).

    2 Yu, F. X., Chai, T. F., He, H., Hagen, T. & Luo, Y. Thioredoxin-interacting protein (Txnip) gene expression: sensing oxidative phosphorylation status and glycolytic rate. J Biol Chem 285, 25822-25830, doi:10.1074/jbc.M110.108290 (2010).

    3 Wischhusen, J., Melero, I. & Fridman, W. H. Growth/Differentiation Factor-15 (GDF-15): From Biomarker to Novel Targetable Immune Checkpoint. Front Immunol 11, 951, doi:10.3389/fimmu.2020.00951 (2020).

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    Referee #3

    Evidence, reproducibility and clarity

    In this well-written manuscript, the authors show that chemotherapy increases a MondoA-dependent oxidative stress-associated protein, TXNIP, in chemotherapy-responsive colorectal cancer cells. They show that TXNIP negatively regulates GDF-15 expression. GDF-15, in turn, correlates with the presence of T cells (Treg), and inhibits CD4 and CD8 T cell stimulation. In advanced disease and chemo-resistant cancers, upregulation of TXNIP and downregulation of GDF-15 appear to get lost. Based on a somewhat smallish data set, the authors suggest that the pre-treatment GDF-15/TXNIP ratio can predict responses to oxaliplatin treatment. This is a very interesting, novel finding. In general, the quality of the experiments and the data are high and the conclusions appear sound. Still, there are a number of aspects that should still be improved:

    The observed loss of the ROS - MondoA - TXNIP - GDF15 axis in chemoresistant and/or metastatic tumors implies that another transcription factor or pathway becomes dominant upon tumor progression. As this switch would be key to better understanding the mechanism underlying the prognostic role of the TXNIP/GDF15 ratio, the authors should at least do data mining followed by ChEA or Encode (or other) analysis to identify transcription factors or pathways that become activated in late-stage/metastatic CRC cells. There is a high likelihood that a transcription factor or pathway involved in GDF-15 upregulation in cancer (e.g. p53, HIF1alpha, Nrf2, NF-kB, MITF, C/EBPß, BRAF, PI3K/AKT, MAPK p38, EGR1) supersedes the inhibitory effect of the MondoA-TXNIP axis. As it stands, the proposed loss of function of the ROS - MondoA - TXNIP - GDF-15 axis is far less convincing than almost all other aspects of the study.

    My further criticisms are mostly more technical:

    Figure 2 I-L: What was the extent of MondoA downregulation achieved by siRNA treatment? Could the effects also be seen with the small molecule mondoA inhibitor SBI-477 (or a related substance)?

    How do you explain the different GDF-15 levels between untreated non-target control cells (NTC) and TXNIP knock-down cells (TKO) in Figures 3C-F?

    In figures 3 E-G the dots for the individual measurements should be indicated. This would be more informative than just the bar graphs.

    Figure 4C,D and Table 3: Data on the role of GDF-15 in CRC are largely valedictory of previous work (e.g. Brown et al. Clin Cancer Res 2003, 9(7):2642-2650, Wallin et al., Br J Cancer. 2011 May, 10;104(10):1619-27). Therefore, the previous studies should be cited.

    Figure 5C-F: Please indicate in the figure legend how proliferation was assessed.

    Figure S8E-G: Please indicate the analysed parameters in the graphs. In Figure S8G, the legend just indicates that "aggression of tumour" is dichotomized and plotted. This clearly requires a better definition.

    The authors propose a novel ROS - MondoA - TXNIP - GDF15 - Treg axis, where MondoA activation, TXNIP up- and GDF-15 downregulation enhance tumor immunogenicity. While this axis has been analyzed in some detail, GDF-15 is not only linked to induction of regulatory T cells. There has been a report showing that GDF-15/MIC-1 expression in colorectal cancer correlates with the absence of immune cell infiltration (Brown et al. Clin Cancer Res 2003, 9(7):2642-2650). The link between GDF-15 and immune cell exclusion has also been confirmed in other conditions, including different cancers (Kempf et al. Nat Med 2011, 17(5):581-588, Roth P et al. Clin Cancer Res 2010, 16(15):3851-3859, Haake et al. Nat Commun 2023, 14(1):4253). A key mechanism is the GDF-15 mediated inhibition of LFA-1 activation on immune cells. As the authors argue that the described pathways turns cold tumors hot in response to oxaliplatin-based chemotherapy, this GDF-15 dependent immune cell exclusion mechanism might be at least as relevant than induction of Treg. Likewise, inhibition of dendritic cell maturation by GDF-15 (Zhou et al. PLoS One 2013, 8(11):e78618) could explain why GDF-15high tumors are immunologically cold.

    The authors propose that the pathways discovered by them contributed to the "heating up" of the tumor microenvironment after oxalilatin-based chemotherapy. The authors should thus look in their data sets for the presence of cytotoxic T cells and their possible correlation with TXNIP and GDF-15 levels.

    The paragraph on GDF-15 receptors needs to be corrected: The purported role of a type 2 transforming growth factor (TGF)-beta receptor in GDF-15 signalling had been due to a frequent contamination of recombinant GDF-15 with TGF-beta (Olsen et al. PLoS One 2017, 12(11):e0187349). There have been a number of screenings for GDF-15 receptors that have all failed to show an interaction between GDF-15 and TGF-beta receptors. Instead, only GFRAL was found in these large-scale screenings (Emmerson et al. Nat Med 2017, 23(10):1215-1219, Hsu et al. Nature 2017, 550(7675):255-259, Mullican et al. Nat Med 2017, 23(10):1150-1157, Yang et al. Nat Med 2017, 23(10):1158-1166). The one subsequent report that shows a link between GDF-15, engagement of CD48 on T cells and induction of a regulatory phenotype (Wang et al. J Immunother Cancer 2021, 9(9)) still awaits independent validation. Considering that CD48 lacks an intracellular signaling domain that would be critical for a classical receptor function, I recommend to be more cautious regarding the role of CD48 as GDF-15 receptor. Given the mechanism outlined by Wang et al. the word interaction partner might be more apt. Moreover, an anti-GDF-15 antibody would be a good control for the experiments involving an anti-CD48 antibody in Figure 5.

    Cell surface externalization of annexin A1 has been described as a failsafe mechanism to prevent inflammatory responses during secondary necrosis (PMID: 20007579). Thus, I am surprised that the authors list annexin A1 among the immune-stimulatory molecules exposed or released in response to chemotherapy-induced cell death (line 103). Please clarify!

    Referee Cross-Commenting

    Regarding the cross-comment by referee 2: In my opinion, the data shown in Figure 3C-H clearly demonstrates that TXNIP can repress GDF-15 expression. I agree that there will likely be further regulators. The GDF-15 promoter is constantly regulated by a multitude of factors (which mostly induce transcription). As downregulation of GDF-15 in response to oxaliplatin is the opposite of the frequently described induction of GDF-15 upon chemotherapy, net effects may always be "smudged" by contributions from different pathways (e.g. by cell stress due to siRNA transfection). Therefore, I believe that the data are as good as it will get. Accordingly, I would not force the authors to further amplify the observed effect.

    cross comment regarding referee #1: I share the general assessment of the referee and recognize the very detailed mechanistic analysis. To further support the moderate effects of the MondoA knockdown, a small molecule inhibitor like SBI-477 might be useful. (I had already suggested using this inhibitor to support these data.) Still, my view on the potential relevance of oxaliplatin-induced, TXNIP-independent downregulation of GDF-15 differs from that of referee 1. In the clinics, platinum-based chemotherapy is one of the strongest inducers of GDF-15 (compare Breen et al. GDF-15 Neutralization Alleviates Platinum-Based Chemotherapy-Induced Emesis, Anorexia, and Weight Loss in Mice and Nonhuman Primates. Cell Metabolism 32(6), P938-950, 2020.DOI:https://doi.org/10.1016/j.cmet.2020.10.023). I was thus surprised that the authors found a pathway, which leads to an outcome that an exactly opposite effect. Thus far, the only obvious reason for reduced GDF-15 secretion upon treatment with cytotoxic drugs was a reduction in tumor cell number due to cytotoxicity. Still, the authors managed to convince me that the described pathway (ROS - MondoA - TXNIP - GDF-15) exists. (Here, I still largely concur with referee 1.) Moreover, as we have identified some factors required for GDF-15 biosynthesis that could easily interact with TXNIP, I find the proposed mechanism plausible. Nevertheless, as a downregulation of GDF-15 in response to chemotherapy is hardly ever observed in late-stage cancers, I believe that the observed switch in pathway activation between early- and late-stage cancers might be highly relevant - in particular, as there is so much evidence for platinum-based induction of GDF-15 in late-stage cancer patients. Emphasizing the divergent clinical observations (e.g. by Breen et al.) could thus help to put the finding into perspective. Analysing TXNIP-independent mechanisms involved in the oxaliplatin-dependent repression of GDF-15, as suggested by referee #1, will require enormous efforts and resources, and may still turn out to be fruitless. Personally, I would thus be content if the authors just mentioned possible contributions from other pathways upon cancer progression. To me, the described pathway seems to be limited to early-stage cancers, and the actual finding that GDF-15 is downregulated is an interesting observation, irrespective of further involved pathways.

    cross comment regarding referee #2: I fully agree with the referee that activation of the pathway by further chemotherapeutic drugs could be a valuable addition. As Guido Kroemer´s lab has described oxaliplatin to induce a more immunogenic cell death compared to other platinum-based chemotherapies, even a rather limited comparison between oxaliplatin and cisplatin could be very interesting.

    Significance

    In general, this is a very interesting manuscript describing a cascade of events that may contribute to successful chemotherapy (which likely requires induction of an immune response against dying tumor cells.) The observation that this pathway is only active in early/non-metastatic cancer cells is striking. Unfortunately, the authors cannot explain inactivation of this pathway in later stage/ metastatic/ highly aggressive cancers. Understanding this switch could easily be the most important finding triggered by this report. Therefore, I highly recommend to make some effort in this direction. Strikingly, the authors find that disruption of TXNIP-mediated GDF-15 downregulation is strongly associated with worse prognosis. They also suggest that this ratio could indicate whether a patient will respond to oxaliplatin-based chemotherapy.

    Altogether, the findings described in manuscript are very novel and may have prognostic (or, in case of the presumed loss of the MondoA - TXNIP - GDF-15 pathway) therapeutic implications. Thus, the manuscript certainly fills various gaps and should be of major interest for cell biologists working on immunogenic cell death, or colorectal cancer, or MondoA, TXNIP or GDF-15. Still, due to its translational implications, it would also be worthwhile reading for a large number of researchers in the oncology field.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Deng et al. investigates a mechanistic link between TXNIP and GDF15 expression and oxaliplatin treatment and acquired resistance. They observe an upregulation in TXNIP expression in the tumors of patients who have previously received chemotherapy. They demonstrate oxaliplatin-driven MondoA transcriptional activity is what underlies the induction of TXNIP. They further demonstrate that TXNIP is a negative regulator of GDF15 expression. Together, oxaliplatin induces MondoA activity and TXNIP expression, resulting in a downregulation of GDF15 expression and consequently decreased Treg differentiation.

    Major Comments

    1. The authors suggest that TXNIP induction and GDF15 downregulation are a common effect of chemotherapies; however, the mechanistic studies were limited to oxaliplatin. The authors should clarify this point through further investigation using other commonly used CRC chemotherapies (5-FU, irinotecan, etc.), or through textual changes. To be clear, I think that the oxaliplatin results could potentially stand on their own but would require additional clarification. For example, regarding the patient samples analyzed in 1D and 4F, which patients received oxaliplatin? Could the analysis of publicly available molecular data be drilled down to just the patients who received oxaliplatin?
    2. The data demonstrating the induction of MondoA transcriptional activity and TXNIP expression in response to oxaliplatin treatment is quite convincing. The data regarding ROS induction of TXNIP is interesting, especially in light of other studies arguing that ROS limits MondoA activity (PMID: 25332233). Given this apparent disparity, I think that this study could really be strengthened by also investigating other potential mechanisms of oxaliplatin induction of MondoA. In particular, given many studies arguing for direct nutrient-regulation of MondoA, the authors should address the potential for oxaliplatin regulation of glucose availability and a potential glucose dependence of oxaliplatin-induced TXNIP.
    3. In line with the previous point, since MondoA activity and TXNIP expression are sensitive to glucose levels, the authors should investigate oxaliplatin-regulation of TXNIP under physiological glucose levels. No need to replicate everything, just key experiments.
    4. The authors did a good job of linking TXNIP and GDF15 in untreated conditions; however, the data arguing for oxaliplatin regulation of GDF15 through TXNIP is less clear. For example, in 3B-H, oxaliplatin treatment reduces GDF15 approximately to the same extent in the NTC and TKO cells, potentially in line with a mechanism of downregulation that doesn't involve TXNIP.

    Minor Comments

    1. The presentation of data in Figure 5 is confusing. A-B include raw cell numbers, whereas C-F show "normalized proliferation." What does this mean? And how was the normalization done?

    Referee Cross-Commenting

    cross-comment regarding reviewer #1

    I agree with the referee that the link between TXNIP and GDF15 is weak, though as I mentioned before, this is particularly true in the context of oxaliplatin-regulation of TXNIP. I agree that given all the presented data, it is likely that oxaliplatin-regulation of TXNIP and GDF15 are independent. In my opinion, the referee brought up all valid concerns, but this is by far the biggest concern that I share.

    cross-comment regarding reviewer #3

    The major concern that this referee addresses is whether another transcription factor supersedes the proposed MondoA/TXNIP induction in regulating GDF15 expression in later stage CRC. In my opinion, this another other concerns of the referee are all valid, but still I remain unconvinced that TXNIP induction underlies the oxaliplatin-regulation of GDF15. I think fleshing out that aspect of the study would potentially help the authors tease apart how this potential MondoA-TXNIP-GDF15 axis is dysregulated later in CRC progression.

    Significance

    Generally speaking the experiments are well controlled and the findings are significant and novel. Though the link between MondoA activity and ROS could be strengthened, and the data could be validated under more physiological settings. Further, the authors should clarify their interpretations so as to not overstate the findings.

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    Referee #1

    Evidence, reproducibility and clarity

    This is well done and interesting paper examining the connection between TXNIP and GDF15. The main thrust is that TXNIP upregulation chemotherapies, such as Oxa, results in an a down regulation of GDF15 early in tumorigenesis. Later in tumorigenesis, TXNIP upregulation is less pronounced, elevating GFP15 resulting in a blockage of tumor suppressive immune responses. Generally the work is convincing. For example, it's clear that TXNIP is up regulated by Oxa in an ROS and MondoA-dependent manner. Likewise its quite clear TXNIP loss reads to an upregulation of GDF15. However, it's also quite clear that Oxa suppresses GDF15 in a manner that appears to be completely independent of TXNIP. The writing in the paper implies strongly that there is a mechanistic connection between TXNIP and GDF15, but no experiments investigate this possibility. It seems equally likely that TXNIP and GDF15 represent independent parallel pathways. Even if TXNIP is a direct regulator of GDF15, it's also clear that other "factors" up or down-regulated by Oxa also contribute to the regulation of GDF15. These are not explored and even though TXNIP is highly regulated genes shown Figure 2 that are not identified or discussed that may also be contributing to GDF15 regulation. Further, the experiments treating PBMCs with conditioned media contain other cytokines/factors, in addition to GDF15, that likely also contribute the observed effects on the different immune cells understudy. The conditioned media from GDF15 knock out cells are a good experiment, but the media is not rigorously tested to see what other cytokines/factors might have also been depleted. Perhaps a GDF15 complementation experiment would help here. Finally, even if completely independent, a TXNIP/GDF15 ratio does seem to have utility in determining chemo-therapeutic response.

    Other major points:

    1. Please label the other highly regulated genes shown in Fig 2A and B. Might they also explain some of the underlying biology. This could be on the current figures or in a supplement, though the former is preferred.
    2. Please address why the TXNIP induction is so much less in patient-derived organoids vs. cell line spheroids (Fig S2). By the western blots, TXNIP inductions in the organoids looks quite modest. Further, the text is quite cryptic and implies that the "upregulation" is similar in both organoids and spheroids.
    3. What was the rationale of performing the MS experiment on control and TXNIP KO DLD1 cells in the absence of oxaliplatin? The other experiments in Fig 3 clearly show that Oxa can repress GDF15 even in the absence of TXNIP, which implicates other pathways. ARRDC4? Or something else? This needs to be addressed.
    4. The data in 3J with the MondoA knockdown is not convincing. The knockdown is weak and TXNIP goes down a smidge. Agree that GDF15 goes up

    Minor points

    1. Line 79. The "loss" of TXNIP/GDF15 axis is confusing. It's really loss of TXNIP and upregulation of GDF15, right?
    2. Please provide an explanation for the different stages in tables 1 and 2. This will likely not be clear to non-clinicians.
    3. Line 231 should probably read ...cysteine (NAC), a reactive oxygen species inhibitor,
    4. Line 247, should be RT-qPCR I think.
    5. Lines 343-345. I don't quite understand the wording. Does this mean to say that 675 soluble proteins were not changed between the condition media from both cell populations?
    6. The data in FigS1 B and C don't seem to reach the standard p value of > 0.05

    Referee Cross-Commenting

    cross comment regarding referees 2 and 3 above. I'm am convinced that TXNIP is at least contemporaneously upregulated with GDF15 dowregulation. However, the strong implication from the writing is that TXNIP regulates GDF15 directly. I agree with the comment above that exploring mechanisms may be open-ended especially as TXNIP has been implicated in gene regulation by several different mechanism. I'd be satisfied with a more open-minded discussion of potential mechanisms by which TXNIP may repress GDF15 and the possibility of other parallel pathways that likely contribute to GDF15 repression.

    Significance

    This is an interesting contribution but the mechanistic connection between GDF15 and TXNIP is relatively weak. That said, even as independent variables they do seem to have utility in predicting therapeutic response.

  5. Version published to 10.1101/2023.08.10.552749v1 on bioRxiv