Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

The role of telomeres in sustained tumor growth is well understood. However, mechanisms of how telomeres might impact the tumor microenvironment (TME) are not clear. Upon examining tumor associated macrophages (TAMs) in 94 hormone-negative (triple-negative) breast cancer (TNBC) cases we found infiltration of TAMs to be telomere sensitive: Tumors with relatively short telomeres had higher abundance of TAM and vice versa. This observation was replicated across TNBC clinical tissue, patient-derived organoids, tumor xenografts and cancer cells with long/short telomeres. Mechanistically, we demonstrate that non-telomeric binding of TRF2, a telomere-repeat-binding-factor; at the interleukin receptor IL1R1 promoter directly activates IL1R1 through recruitment of the histone-acetyl-transferase p300 and consequent H3K27 acetylation. Interleukin-1signaling could be induced in TRF2-high cells through ligands IL1A/B, but not TNFα, and abrogated by the receptor antagonist IL1RA, supporting specificity of the TRF2-IL1R1axis. TRF2 binding at the IL1R1 promoter was mediated by G-quadruplex motifs and was sensitive to telomere length – thereby establishing telomere-length-dependent regulation of IL1R1 and IL1-mediated TAM infiltration in cancers. Our results reveal a heretofore unknown function of telomeres in interleukin signaling and anti- tumor immune response, through non-telomeric TRF2. Therefore, we propose telomere length as a novel biomarker underlying patient-specific response to cancer immunotherapy.

Mutant TP53 prevents Telomere Shortening in Acute Myeloid Leukemia

Background High telomerase activity represents a critical feature of hematopoietic stem cells. Excessive shortening of telomere length (TL) due to replicative stress may be - in analogy to many solid tumors - a hallmark of myeloid neoplasia (MN). Also, telomeric footprints in leukemic genomes may vary between various subtypes corresponding to the differentiation arrest at various stages of hematopoietic ontogeny or specific molecular defects. Critical TL shortening has been associated with genomic instability and accelerated acquisition of genomic lesions leading to a more aggressive phenotype. These processes have not been systematically studied in MN, especially AML. Aim By taking advantage of next-generation sequencing to assay both molecular features and TL within large cohorts of patients, we tested the hypothesis that TL shortening is excessive in highly proliferative MN, but that distinct invariant differences characterize genetic subtypes. Methods Our cohort included AML (N=734), MDS (N=701), healthy controls (HC) (N=11) and PNH (N=102) serving as clonal non-malignant controls. All patients were diagnosed according to WHO standards before being subjected to transcriptome (WTS) and genome (WGS, 100x) sequencing. To retrieve TL characteristics and telomere repeat heterogeneity from WGS data, we used TelomereHunter (TH). In parallel, we performed C-Circle assays. Patients were annotated for clinical features and analyzed for genetic/transcriptomic patterns. Results For a subset of patients for whom corresponding benign lymphocyte DNA was available a significant TL shortening in blasts vs control lymphocytes (A; P=.0023) was detected. While age correlation was established in controls, despite a trend, in MN age did not significantly affect TL (B) and thus subsequent comparisons were not adjusted for age. Next, we studied a cohort of patients with AML, MDS, PNH and HC and found that TL shortening was an overarching finding in AML, MDS and PNH as compared to HC (C). Since no matched DNA was available as reference, we examined the distribution of TL across different age cohorts, AML patients divided according to age cohorts harbored TL in a similar range (D, P=.057). Classic morphologic (E) or cytogenetic subtypes AML exhibited no difference. Similarly, no differences were found between high and low risk MDS patients (not shown). The variability of TL ranges suggested that there may be molecular factors which affect individual TL. When we compared TL grouped according to frequent mutations, only TP53 mutations were associated with longer TL (F, P<.0001). A significant positive correlation (G, P=.021) between TL and TP53 clonal burden was found; samples with the longest vs shortest TL showed significantly higher TP53 VAF (H, P=.0229). In analogy, the presence of multiple TP53 mutations (putative biallelic inactivation) showed longer TL than single hits but no association was found between the nature of mutations and TL (I, J, K). Availability of WTS data allowed us to assess the telomerase activity using the EXTEND score (ES) which has been shown to assess telomerase activity. Indeed, the ES was correlated with TL (L) and TP53 mutant status was associated with a higher ES compared to WT samples (M, P<.0001). Similarly, because of the compensatory upregulation of TP53 in mutant cases, we have also found that TP53 mRNA levels correlated with ES (N P<.0001). Another explanation of TL increase could be the occurrence of alternative lengthening (ALT). TH software allows for estimation of the abundance of specific telomeric repeats. Singleton analysis showed that increase in telomere repeats variants (TTTGGG, O, P=.003) was related to mutations in TP53 arguing against the involvement of ALT. The final confirmation that TL extension was not due to ALT was provided by C-Circle assays. When C-Circle assays were performed for samples with a high/low TL and mutant/WT TP53, none of the subgroups was identified as ALT + (P). Conclusion We stipulate that TL measurements using NGS will be helpful to investigate pathophysiological features associated with TL shortening. Availability of therapies targeting the telomere machinery (Imetelstat) may offer an opportunity for personalized therapy beyond MPN, its current indication. It remains to be tested whether long TL associated with TP53 mutations can serve as marker of sensitivity or resistance to these agents. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy; Novartis: Consultancy.

The Intra- and Extra-Telomeric Role of TRF2 in the DNA Damage Response

Telomere repeat binding factor 2 (TRF2) has a well-known function at the telomeres, which acts to protect the telomere end from being recognized as a DNA break or from unwanted recombination. This protection mechanism prevents DNA instability from mutation and subsequent severe diseases caused by the changes in DNA, such as cancer. Since TRF2 actively inhibits the DNA damage response factors from recognizing the telomere end as a DNA break, many more studies have also shown its interactions outside of the telomeres. However, very little has been discovered on the mechanisms involved in these interactions. This review aims to discuss the known function of TRF2 and its interaction with the DNA damage response (DDR) factors at both telomeric and non-telomeric regions. In this review, we will summarize recent progress and findings on the interactions between TRF2 and DDR factors at telomeres and outside of telomeres.

Targeting chromosome trisomy for chromosome editing

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

Analysis of Ustilago maydis pot1 reveals context-dependent, dichotomous regulation of homology-directed DNA repair factors by a telomere protein

The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the biochemical activity and genetic mechanisms of Pot1 in Ustilago maydis, a fungal model that recapitulates key features of mammalian telomere regulation. We found that U. maydis Pot1 binds directly to Rad51 and regulates the latter’s strand exchange activity. Deleting an N-terminal domain of Pot1 implicated in Rad51-binding caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere replication. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1-deficient cells were suppressed, and cell viability was rescued by the deletion of rad51 or brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection.

Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

Targeting Chromosome Trisomy for Chromosome Editing

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection makers. Upon construction of cells carrying makers targeted in one of the trisomic chromosome 2s, cells that have lost makers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes. (197 words)

RTEL1 influences the abundance and localization of TERRA RNA

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.