Telomere length sensitive regulation of Interleukin Receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

Reviewer #1 (Public Review):

Summary:
This manuscript from Mukherjee et al examines potential connections between telomere length and tumor immune responses. This examination is based on the premise that telomeres and tumor immunity have each been shown to play separate, but important, roles in cancer progression and prognosis as well as prior correlative findings between telomere length and immunity. In keeping with a potential connection between telomere length and tumor immunity, the authors find that long telomere length is associated with reduced expression of the cytokine receptor IL1R1. Long telomere length is also associated with reduced TRF2 occupancy at the putative IL1R1 promoter. These observations lead the authors towards a model in which reduced telomere occupancy of TRF2 - due to telomere shortening - promotes IL1R1 transcription via recruitment of the p300 histone acetyltransferase. This model is based on earlier studies from this group (i.e. Mukherjee et al., 2019) which first proposed that telomere length can influence gene expression by enabling TRF2 binding and gene transactivation at telomere-distal sites. Further mechanistic work suggests that G-quadruplexes are important for TRF2 binding to IL1R1 promoter and that TRF2 acetylation is necessary for p300 recruitment. Complementary studies in human triple-negative breast cancer cells add potential clinical relevance but do not possess a direct connection to the proposed model. Overall, the article presents several interesting observations, but disconnection across central elements of the model and the marginal degree of the data leave open significant uncertainty regarding the conclusions.

Strengths:
Many of the key results are examined across multiple cell models.

The authors propose a highly innovative model to explain their results.

Weaknesses:
Although the authors attempt to replicate most key results across multiple models, the results are often marginal or appear to lack statistical significance. For example, the reduction in IL1R1 protein levels observed in HT1080 cells that possess long telomeres relative to HT1080 short telomere cells appears to be modest (Supplementary Figure 1I). Associated changes in IL1R1 mRNA levels are similarly modest.

Related to the point above, a lack of strong functional studies leaves an open question as to whether observed changes in IL1R1 expression across telomere short/long cancer cells are biologically meaningful.

Statistical significance is described sporadically throughout the paper. Most major trends hold, but the statistical significance of the results is often unclear. For example, Figure 1A uses a statistical test to show statistically significant increases in TRF2 occupancy at the IL1R1 promoter in short telomere HT1080 relative to long telomere HT1080. However, similar experiments (i.e. Figure 2B, Figure 4A - D) lack statistical tests.

Reviewer #2 (Public Review):

This study highlights the role of telomeres in modulating IL-1 signaling and tumor immunity. The authors demonstrate a strong correlation between telomere length and IL-1 signaling by analyzing TNBC patient samples and tumor-derived organoids. Mechanistic insights revealed non-telomeric TRF2 binding at the IL-1R1. The observed effects on NF-kB signaling and subsequent alterations in cytokine expression contribute significantly to our understanding of the complex interplay between telomeres and the tumor microenvironment. Furthermore, the study reports that the length of telomeres and IL-1R1 expression is associated with TAM enrichment. However, the manuscript lacks in-depth mechanistic insights into how telomere length affects IL-1R1 expression. Overall, this work broadens our understanding of telomere biology.

Reviewer #3 (Public Review):

Summary:
In this manuscript, entitled "Telomere length sensitive regulation of Interleukin Receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment", Dr. Mukherjee and colleagues pointed out clarifying the extra-telomeric role of TRF2 in regulating IL1R1 expression with consequent impact on TAMs tumor-infiltration.

Strengths:
Upon careful manuscript evaluation, I feel that the presented story is undoubtedly well conceived. At the technical level, experiments have been properly performed and the obtained results support the authors' conclusions.

Weaknesses:
Unfortunately, the covered topic is not particularly novel. In detail, the TRF2 capability of binding extratelomeric foci in cells with short telomeres has been well demonstrated in a previous work published by the same research group. The capability of TRF2 to regulate gene expression is well-known, the capability of TRF2 to interact with p300 has been already demonstrated and, finally, the capability of TRF2 to regulate TAMs infiltration (that is the effective novelty of the manuscript) appears as an obvious consequence of IL1R1 modulation (this is probably due to the current manuscript organization).

Author Response

Reviewer #1 (Public Review):

Summary:

This manuscript from Mukherjee et al examines potential connections between telomere length and tumor immune responses. This examination is based on the premise that telomeres and tumor immunity have each been shown to play separate, but important, roles in cancer progression and prognosis as well as prior correlative findings between telomere length and immunity. In keeping with a potential connection between telomere length and tumor immunity, the authors find that long telomere length is associated with reduced expression of the cytokine receptor IL1R1. Long telomere length is also associated with reduced TRF2 occupancy at the putative IL1R1 promoter. These observations lead the authors towards a model in which reduced telomere occupancy of TRF2 - due to telomere shortening - promotes IL1R1 transcription via recruitment of the p300 histone acetyltransferase. This model is based on earlier studies from this group (i.e. Mukherjee et al., 2019) which first proposed that telomere length can influence gene expression by enabling TRF2 binding and gene transactivation at telomere-distal sites. Further mechanistic work suggests that G-quadruplexes are important for TRF2 binding to IL1R1 promoter and that TRF2 acetylation is necessary for p300 recruitment. Complementary studies in human triple-negative breast cancer cells add potential clinical relevance but do not possess a direct connection to the proposed model. Overall, the article presents several interesting observations, but disconnection across central elements of the model and the marginal degree of the data leave open significant uncertainty regarding the conclusions.

Strengths:

Many of the key results are examined across multiple cell models.

The authors propose a highly innovative model to explain their results.

Weaknesses:

Although the authors attempt to replicate most key results across multiple models, the results are often marginal or appear to lack statistical significance. For example, the reduction in IL1R1 protein levels observed in HT1080 cells that possess long telomeres relative to HT1080 short telomere cells appears to be modest (Supplementary Figure 1I). Associated changes in IL1R1 mRNA levels are similarly modest.

Related to the point above, a lack of strong functional studies leaves an open question as to whether observed changes in IL1R1 expression across telomere short/long cancer cells are biologically meaningful.

Statistical significance is described sporadically throughout the paper. Most major trends hold, but the statistical significance of the results is often unclear. For example, Figure 1A uses a statistical test to show statistically significant increases in TRF2 occupancy at the IL1R1 promoter in short telomere HT1080 relative to long telomere HT1080. However, similar experiments (i.e. Figure 2B, Figure 4A - D) lack statistical tests.

TRF2 overexpression resulted in ~ 5-fold or more change in IL1R1 expression. Compared to this, telomere length-dependent alterations in IL1R1 expression, although about 2-fold, appear modest (~ 50% reduction in cells with long telomeres across different model systems used). Notably, this was consistent and significant across cell-based model systems and xenograft tumors (see Figure 1). Unlike TRF2 induction, telomere elongation or shortening vary within the permissible physiological limits of cells. This is likely to result in the observed variation in IL1R1 levels. For biological relevance, we further demonstrated that IL1 signalling in TNBC tissue and tumor organoids, and M2 macrophage infiltration, was significantly dependent on telomere length. Details of tests of significance were included in the individual figure legends. Based on the comment here we will expand on it in a dedicated paragraph in the methods section to make the information clearer for readers. We noticed that the stars (*) denoting statistical significance were omitted in some ChIP-experiment figures. This was likely an error during figure assembly for PDF conversion. We thank the reviewer for bringing this up; necessary changes will be made in the revised manuscript.

Reviewer #2 (Public Review):

This study highlights the role of telomeres in modulating IL-1 signaling and tumor immunity. The authors demonstrate a strong correlation between telomere length and IL-1 signaling by analyzing TNBC patient samples and tumor-derived organoids. Mechanistic insights revealed non-telomeric TRF2 binding at the IL-1R1. The observed effects on NF-kB signaling and subsequent alterations in cytokine expression contribute significantly to our understanding of the complex interplay between telomeres and the tumor microenvironment. Furthermore, the study reports that the length of telomeres and IL-1R1 expression is associated with TAM enrichment. However, the manuscript lacks in-depth mechanistic insights into how telomere length affects IL-1R1 expression. Overall, this work broadens our understanding of telomere biology.

The mechanism of how telomere length affects IL1R1 expression involves sequestration and reallocation of TRF2 between telomeres and gene promoters (in this case, the IL1R1 promoter). We have previously shown this across multiple genomic sites (Mukherjee et al, 2018; reviewed in J. Biol. Chem. 2020, Trends in Genetics 2023). We have described this in the manuscript along with references citing the previous works. A scheme explaining the model was provided as Additional Supplementary Figure 1, along with a description of the mechanistic model.

Figure 1-4 in main figures describe the molecular mechanism of telomere-dependent IL1R1 activation. This includes ChIP data for TRF2 on the IL1R1 promoter in long/short telomeres, as well as TRF2-mediated histone/p300 recruitment and IL1R1 gene expression. We further show how specific acetylation on TRF2 is crucial for TRF2-mediated IL1R1 regulation (Figure 5).

Reviewer #3 (Public Review):

Summary:

In this manuscript, entitled "Telomere length sensitive regulation of Interleukin Receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment", Dr. Mukherjee and colleagues pointed out clarifying the extra-telomeric role of TRF2 in regulating IL1R1 expression with consequent impact on TAMs tumor-infiltration.

Strengths:

Upon careful manuscript evaluation, I feel that the presented story is undoubtedly well conceived. At the technical level, experiments have been properly performed and the obtained results support the authors' conclusions.

Weaknesses:

Unfortunately, the covered topic is not particularly novel. In detail, the TRF2 capability of binding extratelomeric foci in cells with short telomeres has been well demonstrated in a previous work published by the same research group. The capability of TRF2 to regulate gene expression is well-known, the capability of TRF2 to interact with p300 has been already demonstrated and, finally, the capability of TRF2 to regulate TAMs infiltration (that is the effective novelty of the manuscript) appears as an obvious consequence of IL1R1 modulation (this is probably due to the current manuscript organization).

Here we studied the TRF2-IL1R1 regulatory axis (not reported earlier by us or others) as a case of the telomere sequestration model that we described earlier (Mukherjee et al., 2018; reviewed in J. Biol. Chem. 2020, Trends in Genetics 2023). This manuscript demonstrates the effect of the TRF2-IL1R1 regulation on telomere-sensitive tumor macrophage recruitment. To the best of our knowledge, no previous study connects telomeres of tumor cells mechanistically to the tumor immune microenvironment. Here we focused on the IL1R1 promoter and provided mechanistic evidence for acetylated-TRF2 engaging the HAT p300 for epigenetically altering the promoter. This mechanism of TRF2 mediated activation has not been previously reported. Further, the function of a specific post translational modification (acetylation of the lysine residue 293K) of TRF2 in IL1R1 regulation is described for the first time. Additional experiments showed that TRF2-acetylation mutants, when targeted to the IL1R1 promoter, significantly alter the transcriptional state of the IL1R1 promoter. To our knowledge, the function of any TRF2 residue in transcriptional activation had not been previously described. Taken together, these demonstrate novel insights into the mechanism of TRF2-mediated gene regulation, that is telomere-sensitive, and affects the tumor-immune microenvironment. We are considering the suggestion to reorganize the manuscript to highlight the novel aspects of our work more convincingly.