Telomere-specific Reverse Transcriptase Research Articles

Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells.

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRXKO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRXKO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRXKO TERCmut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. IMPLICATIONS: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.

Abstract 3464: CRISPR-mediated inactivation of ATRX and DAXX in pancreatic neuroendocrine tumor cell lines

Abstract Cancer cells must find a way to subvert replicative senescence in order to achieve cellular immortality. While most malignancies (> 90 %) overcome this critical barrier by reactivating the telomerase enzyme, a telomere-specific reverse transcriptase; other cancers (5 - 10 %) utilize a telomerase-independent pathway of telomere maintenance, referred to as Alternative Lengthening of Telomeres (ALT). ALT is thought to utilize homologous recombination and DNA damage repair (DDR) machinery to maintain the chromosome ends. Importantly, this mechanism appears to be cancer-specific and dependent on alterations in chromatin dynamics at the telomeres, almost invariably facilitated by inactivating mutations in the genes, ATRX and DAXX. The protein products of these genes are responsible for the deposition of histone variant H3.3 at repetitive regions of DNA (e.g. telomeres) and when altered, the affected cells tend to exhibit the ALT phenotype. Pancreatic neuroendocrine tumors (PanNETs) are an example of a tumor type that often employs the ALT pathway with coincident somatic mutations in either the ATRX or DAXX genes. PanNETs are the second most common pancreatic malignancy, with only 40% of affected patients surviving past 10 years. Recently, we and others have demonstrated that in primary PanNETs, ALT is an independent prognostic marker, associated with aggressive clinicopathologic behavior and reduced recurrence-free survival. In addition, current data suggest that ALT-positive cancer cells are sensitive to inhibition of the DNA-damage mediator, ATR. In light of these findings, there seems to be an opportunity to develop ALT specific therapeutic modalities that would have considerable clinical utility. Models of ALT-positive PanNET pathology are needed to further progress in this direction, unfortunately, there are currently no ALT-positive PanNET cell lines. BON1 and QGP1, the two well characterized PanNET cell lines that are available, are both ALT-negative and retain expression of telomerase, ATRX, and DAXX. In an attempt recapitulate the ALT phenotype in a PanNET cell line, we derived a panel of isogenic cell lines from both BON1 and QGP1 by utilizing the CRISPR-Cas9 nickase system to target ATRX and DAXX. In both cell lines, we have successfully established 5 independent subclones with inactivating mutations in either ATRX or DAXX, validated by Sanger sequencing. We are currently characterizing these novel subclones for the hallmarks of ALT, including the presence of ultrabright telomeric foci, dramatic telomere length heterogeneity, and the presence of extrachromosomal telomeric DNA (e.g. c-circles). Once validated, we plan to investigate whether these ATRX or DAXX-deficient subclones are more sensitive to DDR pathway inhibition (i.e. ATR, ATM, CHK1 and CHK2) pharmacologically or with RNA interference in order to identify new therapeutic strategies for PanNETs. Citation Format: Anthony J. Rizzo, Jacqueline A. Brosnan-Cashman, Mindy K. Graham, Alan K. Meeker, Christopher M. Heaphy. CRISPR-mediated inactivation of ATRX and DAXX in pancreatic neuroendocrine tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3464. doi:10.1158/1538-7445.AM2017-3464

Abstract 3412: Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells

Abstract A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by short telomeres. The majority of cancers overcome this critical replication barrier by upregulating the telomerase enzyme, a telomere-specific reverse transcriptase. However, a subset of cancers lack telomerase, and telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is dependent on homologous recombination. Across a variety of tumor types, our laboratory and others have reported a robust correlation between ALT and recurrent cancer-associated somatic inactivating mutations in the ATRX-DAXX chromatin remodeling complex. In a previous comprehensive cancer survey of ALT, we reported that ALT was highly prevalent in some tumor types (e.g. astrocytomas, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive primary prostate cancer (out of 1,176 analyzed). However, we subsequently found a subset of metastatic prostate cancers that harbor ALT, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting ALT, with the explicit purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic targets for men with ALT-positive lethal metastatic prostate cancer. Inactivating mutations in either ATRX or DAXX using the CRISPR/Cas9n system were introduced into the genetically well-characterized, telomerase-positive (ALT-negative) prostate cancer cell lines, LAPC-4 and CWR22Rv1. Resulting mutant subclones were compared to their parental (or empty vector) counterparts. In these new models, abolishing ATRX expression was sufficient to induce the ALT phenotype in both LAPC-4 and CWR22Rv1, as assessed by multiple biomarkers of ALT, including the presence of bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and extrachromosomal telomere c-circles. Interestingly, abolishing DAXX expression induced ALT in only a subset (3/5) of LAPC-4 DAXX KO clones, and a subset (4/19) of CWR22Rv1 DAXX KO clones. We have successfully activated the ALT telomere maintenance phenotype in two prostate cancer cell lines through CRISPR-mediated targeted gene deletion. We are currently utilizing these isogenic cell lines to further characterize and elucidate the underlying biology of cancers harboring ALT, with the goal of pharmacologically targeting the molecular features unique to the ALT phenotype (e.g. ATR inhibition). The identification of ALT-specific drugs may pave the way for the development of new targeted treatments for the subset of men with ALT-positive lethal metastatic prostate cancer, and more broadly, other ALT-positive cancers. ALT is easily detected in clinical tissue samples, and thus would serve as a predictive biomarker for personalized medicine. Citation Format: Mindy K. Graham. Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3412. doi:10.1158/1538-7445.AM2017-3412

Research progress of EB virus reactivation after allogeneic hematopoietic stem cell transplantation

EB virus reactivation is one of the common complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with the fatality rate as 13.8%.Immune depression and chronic inflammation play key roles in EB virus replication.Besides, the mechanism of EB virus lytic replication modulated by telomere specific reverse transcriptase (TERT) have been explained by recent studies. With preemptive therapy, the EB virus related mortality had been decreased, but the time of intervention remains unclear. Rituximab and early reduction of immunosuppressive therapy are recommended as first-line treatment. Immunotherapy has achieved enormous progress as well. This article reviews literatures on the life cycle, risk factors, diagnosis and treatment of EB virus reactivation. Key words: Epstein-Barr virus infections; Hematopoietic stem cell transplantation; Immunosuppressive agents; Immunotherapy

Abstract 4767: Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism

Abstract A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by telomere shortening. The majority of cancers overcome this critical barrier by up-regulating the enzyme telomerase, a telomere-specific reverse transcriptase. However, for a subset of cancers that lack telomerase, telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is thought to be dependent on homologous recombination. In a variety of tumor types, our laboratory and others have reported a strong correlation between the ALT phenotype and recurrent cancer-associated somatic inactivating mutations ATRX or DAXX, genes encoding chromatin remodeling proteins. In a previous comprehensive survey of the ALT phenotype in cancer, we reported that the ALT phenotype is highly prevalent in some tumor types (e.g. astrocytoma, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive cases in over 1,000 cases of primary prostate cancer. Notably, however, we found a subset of metastatic prostate cancer harbors the ALT phenotype, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting the ALT phenotype, with the purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic approaches for men with lethal metastatic prostate cancer harboring this phenotype. These novel cell lines were generated by inactivating ATRX using the CRISPR cas9 nickase system in a genetically well characterized prostate cancer cell line that normally expresses telomerase, as well as wild-type ATRX and DAXX. In this new model, abolishing ATRX expression is sufficient to induce the ALT phenotype, as assessed by multiple biomarkers of ALT, including bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and c-circles. Interestingly, compared to the parental line, cells with compromised ATRX function have significantly lower telomerase activity and a five-fold decreased expression of telomerase reverse transcriptase (TERT). Ultimately, we will use these isogenic cell lines to further characterize and elucidate the basic biology of cancers harboring ALT, and pharmacologically target the molecular features unique to the ALT phenotype. The identification of ALT-specific drugs will pave the way for the development of new targeted treatments for a subset of men with lethal metastatic prostate cancer that harbor this unique molecular phenotype, and more broadly, other cancers that share the ALT phenotype in common. Citation Format: Mindy K. Graham, Jacqueline Brosnan-Cashman, Anthony Rizzo, Michael Haffner, Alan Meeker, Christopher Heaphy. Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4767.

Cross talk between EBV and telomerase: the role of TERT and NOTCH2 in the switch of latent/lytic cycle of the virus.

Epstein–Barr virus (EBV)-associated malignancies, as well as lymphoblastoid cell lines (LCLs), obtained in vitro by EBV infection of B cells, express latent viral proteins and maintain their ability to grow indefinitely through inappropriate activation of telomere-specific reverse transcriptase (TERT), the catalytic component of telomerase. Our previous studies demonstrated that high levels of TERT expression in LCLs prevent the activation of EBV lytic cycle, which is instead triggered by TERT silencing. As lytic infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies. BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells, negatively affects the expression of BZLF1, the master regulator of viral lytic cycle. We therefore analyzed the interplay between TERT, NOTCH and BATF in LCLs and found that high levels of endogenous TERT are associated with high NOTCH2 and BATF expression levels. In addition, ectopic expression of TERT in LCLs with low levels of endogenous telomerase was associated with upregulation of NOTCH2 and BATF at both mRNA and protein levels. By contrast, infection of LCLs with retroviral vectors expressing functional NOTCH2 did not alter TERT transcript levels. Luciferase reporter assays, demonstrated that TERT significantly activated NOTCH2 promoter in a dose-dependent manner. We also found that NF-κB pathway is involved in TERT-induced NOTCH2 activation. Lastly, pharmacologic inhibition of NOTCH signaling triggers the EBV lytic cycle, leading to the death of EBV-infected cells. Overall, these results indicate that TERT contributes to preserve EBV latency in B cells mainly through the NOTCH2/BAFT pathway, and suggest that NOTCH2 inhibition may represent an appealing therapeutic strategy against EBV-associated malignancies.

Alternative Splicing of hTERT Exon 7 in AML: Biological Fuction and Prognostic Significance

hTERT, a telomere specific reverse transcriptase with a crucial role in telomerase activity, prevents telomeres shortening, a pro-apototic cellular event which occurs at every round of DNA replication. Its regulation is complex as alternative spliced variants which generate the full-length hTERT transcript (+α+β) and transcripts carrying α and/or β deletions (transdominant negative isoform -α+β, inactive products +α-β and -α-β) have been reported. In AML the prognostic significance of hTERT expression is still debated, even if its over-expression has been correlated with a poor clinical outcome. In contrast, up to now no study has dealt with the frequency and the prognostic significance of variant transcripts.Thus, our study was aimed at estimating hTERT isoforms distribution in a series of de novo AML patients (pts), at establishing whether their expression varied between pts with a normal and a complex chromosome pattern, at estimating their prognostic significance. The ninety-seven de novo AML pts included in the present study came to our observation in the period January 2010 and January 2013. They were thirty-eight females and fifty-nine males; their median age was 59 years (range 18-84). On conventional cytogenetic studies fifty-two, whose median age was 56 years (range 24-84), presented a normal karyotype and forty-five, whose median age was 60 years (range 33-84), presented a complex karyotype (≥3 defects); the two pts groups were comparable by age. According to WHO classification 6 (6.1%) pts were classified as M0/M1, 47 (48.4%) as M2, 38 (39.1%) as M4, 5 (5.1%) as M5 and one as M6. An internal tandem duplication (ITD) of the FLT3 gene and a NPM1 mutation were revealed in 12 and 2 chromosomally normal pts. No CK presented a FLT3 ITD. All pts received standard induction chemotherapy followed by two courses of consolidation treatment. At the time of the study 38 pts achieved a complete remission (CR) and 36 died. Median follow-up was 22.7 months (range: 13.5-60.2).hTERT isoforms expression was determined in bone marrow samples by real-time reverse transcriptase polymerase chain reaction, using SYBR Green I. hTERT transcript (+α+β) primers’ design was made using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), while for the other primers we referred to Capraro et al. 2011. Specific amplifications were confirmed by sequences analysis. In order, to estimate hTERT isoforms expression levels in normal mononuclear cells, twenty-three umbilical cord bloods (UCB) were examined (control group).When the Kruskal-Wallis rank test was applied to compare the expression of hTERT isoforms inAML pts with a normal and a complex karyotype no significant difference was observed (P=0.39, P=0.77; +α+β, -α+β respectively). Instead, independently from karyotype, in Cox univariate analysis, pts who over-expressed the trans-dominant negative isoform (-α+β) experienced a significant better response to treatment than the other pts (P=0.03, HR=2.11; 95% CI:1,13-3,93). In addition, pts who over-expressed inactive products (-α-β) (first group) presented a leukemia-free survival inferior to that of pts who presented a low expression of these products (second group) (P=0,003, HR=3,26; 95% CI:1,33-7,96). Moreover, pts who over-expressed the full-length hTERT transcript (+α+β) and the trans-dominant negative isoform (-α+β) presented only a trend towards significance. The first group of pts presented a worse overall survival (HR=1,66; 95% CI:0.94-2,91), whereas the second group a better overall survival (HR=0,58; 95% CI:0,33-1,02).We conclude that the over-expression of the hTERT trans-dominant negative isoform identifies pts with low-risk AML, whereas the over-expression of the hTERT inactive product (-α-β) identifies pts with high-risk AML. In addition, this last hTERT isoform might control the expression of its trans-dominant negative isoform. Thus, our results suggest an intriguing link between the control of hTERT isoforms expression and AML outcome. DisclosuresNo relevant conflicts of interest to declare.

Telomerase is an independent prognostic marker of overall survival in patients with colorectal cancer

Background:Colorectal cancer (CRC) is an important cause of cancer-related death. Prediction of recurrence is an important issue in the treatment of disease, particularly for stage II patients. The level of telomere-specific reverse transcriptase (hTERT), the catalytic component of the telomerase complex, increases along with CRC progression, but its prognostic value is still unclear.Methods:One hundred and thirty-seven CRC patients were studied for hTERT expression in tumour cells by real-time PCR. hTERT level was evaluated as a prognostic factor of overall survival (OS) in all patients and of disease recurrence in a subgroup of 50 stage II patients.Results:The median hTERT level was 93.8 copies (interquartile range 48–254). Patients with high hTERT levels (above the median) showed a significantly worse survival than those with low hTERT levels (below the median; log-rank test P<0.0001; hazard ratio (HR)=3.30 (95% confidence interval (CI) 1.98–5.52); P<0.0001). The negative prognostic value of high hTERT level is independent of the pathological stage and microsatellite instability (HR=2.09 (95% CI 1.20–3.64), P=0.009). Moreover, in stage II CRC, high hTERT levels identified patients with a higher risk of disease recurrence (HR=3.06 (95% CI 1.03–9.04), P=0.043) and death (HR=3.24 (95% CI 1.37–7.71), P=0.008).Conclusion:hTERT level is an independent prognostic marker of OS in CRC patients. In addition, assessment of hTERT level could improve stratification of stage II CRC patients for the risk of disease recurrence.

Telomere-Specific Reverse Transcriptase (hTERT) and Cell-free RNA in Plasma as Predictors of Pathologic Tumor Response in Rectal Cancer Patients Receiving Neoadjuvant Chemoradiotherapy

To investigate whether the plasma levels of cell-free RNA (cfRNA) and telomere-specific reverse transcriptase mRNA (hTERT) are associated with tumor response in rectal cancer patients who received preoperative chemoradiotherapy (pCRT). Patients who underwent pCRT for rectal cancer and for whom baseline and paired post-pCRT blood samples were available were studied. On the basis of tumor regression score, patients were classified as having response or having no response. Clinical variables and plasma levels of cfRNA and hTERT before and after the pCRT were evaluated. The association between each predictor and tumor response was assessed by univariate and multivariate analyses. Of 98 eligible patients, 45 were determined to respond to therapy, and 53 did not respond to therapy. In univariate analysis, gender (P = 0.040), baseline levels of cfRNA (P = 0.026), post-pCRT levels of both hTERT and cfRNA (P < 0.0001 and P = 0.001, respectively), and the difference between the post- and pre-pCRT levels of both hTERT and cfRNA (P = 0.009 and P = 0.001, respectively) were found to be significant predictors of tumor response. In multivariate analysis, using variables that were available before pCRT, cfRNA levels and gender independently predicted the tumor response, while in multivariate analysis, which used all of the variables available before the surgical procedure, the post-pCRT levels of cfRNA and the difference between the post- and pre-pCRT levels of cfRNA independently predicted tumor response. Plasma levels of cfRNA and hTERT are promising markers of tumor response to pCRT for rectal cancer.

Plasma levels of total RNA and hTERT mRNA as biomarkers of response in rectal cancer patients receiving preoperative chemoradiotherapy.

3648 Background: The levels of telomere-specific reverse transcriptase (hTERT) transcripts have been found to be a marker of colorectal cancer and tumor progression. Aim of the study was to investi...

Yeast telomerase is specialized for C/A-rich RNA templates.

Telomeres, the protective caps of eukaryotic chromosomes, are maintained by the enzyme telomerase. This telomere-specific reverse transcriptase (RT) uses a small region of its RNA subunit as template to synthesize telomeric DNA, which is generally G/T rich in the strand that contains the 3' end. To further our understanding of why telomeres are usually G/T rich, we screened Saccharomyces cerevisiae telomerase RNA (TLC1) libraries with randomized template sequences for complementation of a tlc1 deletion and decapping of existing telomeres. Surprisingly, the vast majority of the 60 000 different mutant telomerase templates tested showed no activity in vivo. This deficiency was not due to impaired assembly with the catalytic subunit (Est2p) nor could it be alleviated by enforced telomerase recruitment to the telomeres. Rather, the mutant templates reduced the nucleotide addition processivity of telomerase. The functional RNA template sequences recovered in our screens preferentially contained two or more consecutive rC nucleotides, reminiscent of the wild-type template. Thus, in contrast to retroviral RTs that can reverse transcribe any RNA sequence into DNA, the budding yeast telomerase RT is specialized for its C-rich RNA template.

Telomeres: Beginning to Understand the End

Telomeres are the protein-DNA structures at the ends of eukaryotic chromosomes. In yeast, and probably most other eukaryotes, telomeres are essential. They allow the cell to distinguish intact from broken chromosomes, protect chromosomes from degradation, and are substrates for novel replication mechanisms. Telomeres are usually replicated by telomerase, a telomere-specific reverse transcriptase, although telomerase-independent mechanisms of telomere maintenance exist. Telomere replication is both cell cycle- and developmentally regulated, and its control is likely to be complex. Because telomere loss causes the kinds of chromosomal changes associated with cancer and aging, an understanding of telomere biology has medical relevance.