Protein SUMOylation promotes cAMP-independent EPAC1 activation.

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

The Effects of Smoking on Telomere Length, Induction of Oncogenic Stress, and Chronic Inflammatory Responses Leading to Aging.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

Shaping DNA damage responses: Therapeutic potential of targeting telomeric proteins and DNA repair factors in cancer.

Shelterin proteins regulate genomic stability by preventing inappropriate DNA damage responses (DDRs) at telomeres. Unprotected telomeres lead to persistent DDR causing cell cycle inhibition, growth arrest, and apoptosis. Cancer cells rely on DDR to protect themselves from DNA lesions and exogenous DNA-damaging agents such as chemotherapy and radiotherapy. Therefore, targeting DDR machinery is a promising strategy to increase the sensitivity of cancer cells to existing cancer therapies. However, the success of these DDR inhibitors depends on other mutations, and over time, patients develop resistance to these therapies. This suggests the need for alternative approaches. One promising strategy is co-inhibiting shelterin proteins with DDR molecules, which would offset cellular fitness in DNA repair in a mutation-independent manner. This review highlights the associations and dependencies of the shelterin complex with the DDR proteins and discusses potential co-inhibition strategies that might improve the therapeutic potential of current inhibitors.

F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

BACKGROUND: Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment. METHODS: We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 (F2RL3) was finally selected for verification after functional and survival analyses of the prognostic genes. RESULTS: F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3-knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation. CONCLUSIONS: F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.

Telomere-lengthening germline variants predispose to a syndromic papillary thyroid cancer subtype.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy. 10% to 15% of individuals show familial clustering with three or more affected members, but the factors underlying this risk are unknown. In a group of recently studied individuals with POT1 pathogenic variants and ultra-long telomere length, PTC was the second most common solid tumor. We tested whether variants in POT1 and four other telomere-maintenance genes associated with familial cancer underlie PTC susceptibility. Among 470 individuals, we identified pathogenic or likely pathogenic variants in three genes encoding telomere-binding proteins: POT1, TINF2, and ACD. They were found in 4.5% and 1.5% of familial and unselected cases, respectively. Individuals harboring these variants had ultra-long telomere length, and 15 of 18 (83%) developed other cancers, of which melanoma, lymphoma, and sarcoma were most common. Among individuals with PTC and melanoma, 22% carried a deleterious germline variant, suggesting that a long telomere syndrome might be clinically recognizable. Successive generations had longer telomere length than their parents and, at times, developed more cancers at younger ages. Tumor sequencing identified a single oncogenic driver, BRAF p.Val600Glu, in 10 of 10 tumors studied, but no telomere-maintenance mechanism, including at the TERT promoter. These data identify a syndromic subset of PTCs with locus heterogeneity and telomere lengthening as a convergent mechanism. They suggest these germline variants lower the threshold to cancer by obviating the need for an acquired telomere-maintenance mechanism in addition to sustaining the longevity of oncogenic mutations.

Upregulation of shelterin and CST genes and longer telomeres are associated with unfavorable prognostic characteristics in prostate cancer.

INTRODUCTION: Search for new clinical biomarkers targets in prostate cancer (PC) is urgent. Telomeres might be one of these targets. Telomeres are the extremities of linear chromosomes, essential for genome stability and control of cell divisions. Telomere homeostasis relies on the proper functioning of shelterin and CST complexes. Telomeric dysfunction and abnormal expression of its components are reported in most cancers and are associated with PC. Despite this, there are only a few studies about the expression of the main telomere complexes and their relationship with PC progression. We aimed to evaluate the role of shelterin (POT1, TRF2, TPP1, TIN2, and RAP1) and CST (CTC1, STN1, and TEN1) genes and telomere length in the progression of PC. METHODS: We evaluated genetic alterations of shelterin and CST by bioinformatics in samples of localized (n = 499) and metastatic castration-resistant PC (n = 444). We also analyzed the expression of the genes using TCGA (localized PC n = 497 and control n = 152) and experimental approaches, with surgical specimens (localized PC n = 81 and BPH n = 10) and metastatic cell lines (LNCaP, DU145, PC3 and PNT2 as control) by real-time PCR. Real-time PCR also determined the telomere length in the same experimental samples. All acquired data were associated with clinical parameters. RESULTS: Genetic alterations are uncommon in PC, but POT1, TIN2, and TEN1 showed significantly more amplifications in the metastatic cancer. Except for CTC1 and TEN1, which are differentially expressed in localized PC samples, we did not detect an expression pattern relative to control and cell lines. Nevertheless, except for TEN1, the upregulation of all genes is associated with a worse prognosis in localized PC. We also found that increased telomere length is associated with disease aggressiveness in localized PC. CONCLUSION: The upregulation of shelterin and CST genes creates an environment that favors telomere elongation, giving selective advantages for localized PC cells to progress to more aggressive stages of the disease.

A POT1 Founder Variant Associated with Early Onset Recurrent Melanoma and Various Solid Malignancies.

POT1 (Protection of Telomeres 1) is a key component of the six-membered shelterin complex that plays a critical role in telomere protection and length regulation. Germline variants in the POT1 gene have been implicated in predisposition to cancer, primarily to melanoma and chronic lymphocytic leukemia (CLL). We report the identification of POT1 p.(I78T), previously ranked with conflicting interpretations of pathogenicity, as a founder pathogenic variant among Ashkenazi Jews (AJs) and describe its unique clinical landscape. A directed database search was conducted for individuals referred for genetic counselling from 2018 to 2023. Demographic, clinical, genetic, and pathological data were collected and analyzed. Eleven carriers, 25 to 67 years old, from ten apparently unrelated families were identified. Carriers had a total of 30 primary malignancies (range 1-6); nine carriers (82%) had recurrent melanoma between the ages of 25 and 63 years, three carriers (27%) had desmoid tumors, three (27%) had papillary thyroid cancer (PTC), and five women (63% of female carriers) had breast cancer between the ages of 44 and 67 years. Additional tumors included CLL; sarcomas; endocrine tumors; prostate, urinary, and colorectal cancers; and colonic polyps. A review of a local exome database yielded an allelic frequency of the variant of 0.06% among all ethnicities and of 0.25% in AJs. A shared haplotype was found in all carriers tested. POT1 p.(I78T) is a founder disease-causing variant associated with early-onset melanoma and additional various solid malignancies with a high tumor burden. We advocate testing for this variant in high-risk patients of AJ descent. The inclusion of POT1 in germline panels for various types of cancer is warranted.

TERT-independent telomere elongation and shelterin dysregulation after pulmonary exposure to stainless-steel welding fume in-vivo.

Telomeres are inert DNA sequences (TTAGGG) at the end of chromosomes that protect genetic information and maintain DNA integrity. Emerging evidence has demonstrated that telomere alteration can be closely related to occupational exposure and the development of various disease conditions, including cancer. However, the functions and underlying molecular mechanisms of telomere alteration and shelterin dysregulation after welding fume exposures have not been broadly defined. In this study, we analyzed telomere length and shelterin complex proteins in peripheral blood mononuclear cells (PBMCs) and in lung tissue recovered from male Sprague-Dawley rats following exposure by intratracheal instillation (ITI) to 2 mg/rat of manual metal arc-stainless steel (MMA-SS) welding fume particulate or saline (vehicle control). PBMCs and lung tissue were harvested at 30 d after instillation. Our study identified telomere elongation and shelterin dysregulation in PBMCs and lung tissue after welding fume exposure. Mechanistically, telomere elongation was independent of telomerase reverse transcriptase (TERT) activation. Collectively, our findings demonstrated that welding fume-induced telomere elongation was (a) TERT-independent and (b) associated with shelterin complex dysregulation. It is possible that an alteration of telomere length and its regulatory proteins may be utilized as predictive biomarkers for various disease conditions after welding fume exposure. This needs further investigation.

Human CST complex restricts excessive PrimPol repriming upon UV induced replication stress by suppressing p21.

DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.

Germline POT1 Variants: A Critical Perspective on POT1 Tumor Predisposition Syndrome.

The Protection of Telomere 1 (POT1) gene was identified as a melanoma predisposition candidate nearly 10 years ago. Thereafter, various cancers have been proposed as associated with germline POT1 variants in the context of the so-called POT1 Predisposition Tumor Syndrome (POT1-TPD). While the key role, and related risks, of the alterations in POT1 in melanoma are established, the correlation between germline POT1 variants and the susceptibility to other cancers partially lacks evidence, due also to the rarity of POT1-TPD. Issues range from the absence of functional or segregation studies to biased datasets or the need for a revised classification of variants. Furthermore, a proposal of a surveillance protocol related to the cancers associated with POT1 pathogenic variants requires reliable data to avoid an excessive, possibly unjustified, burden for POT1 variant carriers. We propose a critical perspective regarding data published over the last 10 years that correlate POT1 variants to various types of cancer, other than cutaneous melanoma, to offer food for thought for the specialists who manage cancer predisposition syndromes and to stimulate a debate on the grey areas that have been exposed.

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

Shedding Light on the Interaction Between Rif1 and Telomeres in Ovarian Cancer.

Ovarian cancer, more precisely high-grade serous ovarian cancer, is one of the most lethal age-independent gynecologic malignancies in women worldwide, regardless of age. There is mounting evidence that there is a link between telomeres and the RIF1 protein and the proliferation of cancer cells. Telomeres are hexameric (TTAGGG) tandem repeats at the tip of chromosomes that shorten as somatic cells divide, limiting cell proliferation and serving as an important barrier in preventing cancer. RIF1 (Replication Time Regulation Factor 1) plays, among other factors, an important role in the regulation of telomere length. Interestingly, RIF1 appears to influence the DNA double-strand break (DSB) repair pathway. However, detailed knowledge regarding the interplay between RIF1 and telomeres and their degree of engagement in epithelial ovarian cancer (EOC) is still elusive, despite the fact that such knowledge could be of relevance in clinical practice to find novel biomarkers. In this review, we provide an update of recent literature to elucidate the relation between telomere biology and the RIF1 protein during the development of ovarian cancer in women.

The Altered Functions of Shelterin Components in ALT Cells.

Telomeres are nucleoprotein complexes that cap the ends of eukaryotic linear chromosomes. Telomeric DNA is bound by shelterin protein complex to prevent telomeric chromosome ends from being recognized as damaged sites for abnormal repair. To overcome the end replication problem, cancer cells mostly preserve their telomeres by reactivating telomerase, but a minority (10-15%) of cancer cells use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). Recent studies have found that shelterin components play an important role in the ALT mechanism. The binding of TRF1, TRF2, and RAP1 to telomeres attenuates ALT activation, while the maintenance of ALT telomere requires TRF1 and TRF2. POT1 and TPP1 can also influence the occurrence of ALT. The elucidation of how shelterin regulates the initiation of ALT remains elusive. This review presents a comprehensive overview of the current findings on the regulation of ALT by shelterin components, aiming to enhance the insight into the altered functions of shelterin components in ALT cells and to identify potential targets for the treatment of ALT tumor cells.

Pif1 Helicase Mediates Remodeling of Protein-Nucleic Acid Complexes by Promoting Dissociation of Sub1 from G-Quadruplex DNA and Cdc13 from G-Rich Single-Stranded DNA.

Pif1 is a molecular motor enzyme that is conserved from yeast to mammals. It translocates on ssDNA with a directional bias (5' → 3') and unwinds duplexes using the energy obtained from ATP hydrolysis. Pif1 is involved in dsDNA break repair, resolution of G-quadruplex (G4) structures, negative regulation of telomeres, and Okazaki fragment maturation. An important property of this helicase is to exert force and disrupt protein-DNA complexes, which may otherwise serve as barriers to various cellular pathways. Previously, Pif1 was reported to displace streptavidin from biotinylated DNA, Rap1 from telomeric DNA, and telomerase from DNA ends. Here, we have investigated the ability of S. cerevisiae Pif1 helicase to disrupt protein barriers from G4 and telomeric sites. Yeast chromatin-associated transcription coactivator Sub1 was characterized as a G4 binding protein. We found evidence for a physical interaction between Pif1 helicase and Sub1 protein. Here, we demonstrate that Pif1 is capable of catalyzing the disruption of Sub1-bound G4 structures in an ATP-dependent manner. We also investigated Pif1-mediated removal of yeast telomere-capping protein Cdc13 from DNA ends. Cdc13 exhibits a high-affinity interaction with an 11-mer derived from the yeast telomere sequence. Our results show that Pif1 uses its translocase activity to enhance the dissociation of this telomere-specific protein from its binding site. The rate of dissociation increased with an increase in the helicase loading site length. Additionally, we examined the biochemical mechanism for Pif1-catalyzed protein displacement by mutating the sequence of the telomeric 11-mer on the 5'-end and the 3'-end. The results support a model whereby Pif1 disrupts Cdc13 from the ssDNA in steps.

POT1a deficiency in mesenchymal niches perturbs B-lymphopoiesis.

Protection of telomeres 1a (POT1a) is a telomere binding protein. A decrease of POT1a is related to myeloid-skewed haematopoiesis with ageing, suggesting that protection of telomeres is essential to sustain multi-potency. Since mesenchymal stem cells (MSCs) are a constituent of the hematopoietic niche in bone marrow, their dysfunction is associated with haematopoietic failure. However, the importance of telomere protection in MSCs has yet to be elucidated. Here, we show that genetic deletion of POT1a in MSCs leads to intracellular accumulation of fatty acids and excessive ROS and DNA damage, resulting in impaired osteogenic-differentiation. Furthermore, MSC-specific POT1a deficient mice exhibited skeletal retardation due to reduction of IL-7 producing bone lining osteoblasts. Single-cell gene expression profiling of bone marrow from POT1a deficient mice revealed that B-lymphopoiesis was selectively impaired. These results demonstrate that bone marrow microenvironments composed of POT1a deficient MSCs fail to support B-lymphopoiesis, which may underpin age-related myeloid-bias in haematopoiesis.

POT1 involved in telomeric DNA damage repair and genomic stability of cervical cancer cells in response to radiation.

Though telomeres play a crucial role in maintaining genomic stability in cancer cells and have emerged as attractive therapeutic targets in anticancer therapy, the relationship between telomere dysfunction and genomic instability induced by irradiation is still unclear. In this study, we identified that protection of telomeres 1 (POT1), a single-stranded DNA (ssDNA)-binding protein, was upregulated in γ-irradiated HeLa cells and in cancer patients who exhibit radiation tolerance. Knockdown of POT1 delayed the repair of radiation-induced telomeric DNA damage which was associated with enhanced H3K9 trimethylation and enhanced the radiosensitivity of HeLa cells. The depletion of POT1 also resulted in significant genomic instability, by showing a significant increase in end-to-end chromosomal fusions, and the formation of anaphase bridges and micronuclei. Furthermore, knockdown of POT1 disturbed telomerase recruitment to telomere, and POT1 could interact with phosphorylated ATM (p-ATM) and POT1 depletion decreased the levels of p-ATM induced by irradiation, suggesting that POT1 could regulate the telomerase recruitment to telomeres to repair irradiation-induced telomeric DNA damage of HeLa cells through interactions with p-ATM. The enhancement of radiosensitivity in cancer cells can be achieved through the combination of POT1 and telomerase inhibitors, presenting a potential approach for radiotherapy in cancer treatment.

TINF2 is a major susceptibility gene in Danish patients with multiple primary melanoma.

TINF2 encodes the TINF2 protein, which is a subunit in the shelterin complex critical for telomere regulation. Three recent studies have associated six truncating germline variants in TINF2 that have previously been associated with a cancer predisposition syndrome (CPS) caused by elongation of the telomeres. This has added TINF2 to the long telomere syndrome genes, together with other telomere maintenance genes such as ACD, POT1, TERF2IP, and TERT. We report a clinical study of 102 Danish patients with multiple primary melanoma (MPM) in which a germline truncating variant in TINF2 (p.(Arg265Ter)) was identified in four unrelated participants. The telomere lengths of three variant carriers were >90% percentile. In a routine diagnostic setting, the variant was identified in two more families, including an additional MPM patient and monozygotic twins with thyroid cancer and other cancer types. A total of 10 individuals from six independent families were confirmed carriers, all with cancer history, predominantly melanoma. Our findings suggest a major role of TINF2 in Danish patients with MPM. In addition to melanoma, other cancers in the six families include thyroid, renal, breast, and sarcoma, supporting a CPS in which melanoma, thyroid cancer, and sarcoma predominate. Further studies are needed to establish the full spectrum of associated cancer types and characterize lifetime cancer risk in carriers.

Pot1b -/- tumors activate G-quadruplex-induced DNA damage to promote telomere hyper-elongation.

Malignant cancers must activate telomere maintenance mechanisms to achieve replicative immortality. Mutations in the human Protection of Telomeres 1 (POT1) gene are frequently detected in cancers with abnormally long telomeres, suggesting that the loss of POT1 function disrupts the regulation of telomere length homeostasis to promote telomere elongation. However, our understanding of the mechanisms leading to elongated telomeres remains incomplete. The mouse genome encodes two POT1 proteins, POT1a and POT1b possessing separation of hPOT1 functions. We performed serial transplantation of Pot1b-/- sarcomas to better understand the role of POT1b in regulating telomere length maintenance. While early-generation Pot1b-/- sarcomas initially possessed shortened telomeres, late-generation Pot1b-/- cells display markedly hyper-elongated telomeres that were recognized as damaged DNA by the Replication Protein A (RPA) complex. The RPA-ATR-dependent DNA damage response at telomeres promotes telomerase recruitment to facilitate telomere hyper-elongation. POT1b, but not POT1a, was able to unfold G-quadruplex present in hyper-elongated telomeres to repress the DNA damage response. Our findings demonstrate that the repression of the RPA-ATR DDR is conserved between POT1b and human POT1, suggesting that similar mechanisms may underly the phenotypes observed in human cancers harboring human POT1 mutations.