ALT-associated PML Bodies Research Articles

Abstract 2717: Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma

Abstract The unlimited proliferative capacity of cancer cells is closely linked to maintenance of their telomeres, which shorten with each cell division in normal cells. Cancer cells are able to maintain telomere length by multiple mechanisms, including activation of telomerase and the recombination based alternative lengthening of telomeres (ALT) pathway. ALT is prevalent in osteosarcoma, with approximately 50% of osteosarcoma cases using ALT for telomere maintenance. Mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 correlate with activation of the ALT pathway in several tumor systems. While loss of ATRX is a frequent event in osteosarcoma tumors, alterations of DAXX have not been reported. We characterized the telomere maintenance mechanisms utilized by 11 osteosarcoma cell lines. Of these, 45% (5/11) were ALT positive and 45% (5/11) were telomerase positive. One cell line possessed features of both telomere maintenance mechanisms. Among ALT positive osteosarcoma cell lines, we observed frequent loss of ATRX expression (4/5) and a previously unreported translocation resulting in disruption of DAXX. The translocation abolishes recruitment of DAXX to nuclear PML bodies and prevents normal DAXX function. By reintroducing full length DAXX, we were able to suppress telomere maintenance by ALT as evidenced by multiple assays including loss of C-circles and ALT-associated PML bodies, thus demonstrating that continued DAXX deficiency is necessary for maintenance of the ALT mechanism. Suppression of ALT by DAXX reintroduction did not result in compensatory activation of telomerase. This first demonstration of ALT suppression by DAXX supports a mechanistic connection between loss of the ATRX/DAXX chromatin remodeling complex and telomere maintenance by ALT. Understanding this relationship may uncover vulnerabilities specific to ALT tumors that could potentially lead to the development of targeted therapies for diverse cancers that depend on the ALT pathway. Citation Format: Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Ogan Abaan, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sean R. Davis, Sven Bilke, Paul S. Meltzer. Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma. [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 2717.

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.

Loss of ATRX and DAXX expression identifies poor prognosis for smooth muscle tumours of uncertain malignant potential and early stage uterine leiomyosarcoma.

Uterine smooth muscle tumours of uncertain malignant potential (STUMP) are diagnostically and clinically challenging. The alternative lengthening of telomeres (ALT) telomere maintenance mechanism is associated with poor survival in soft tissue leiomyosarcoma. Time to first recurrence and survival were known for 18 STUMP and 43 leiomyosarcomata (LMS). These were screened for ALT telomere maintenance by the presence of ALT‐associated PML bodies (APBs) and for changes associated with the ALT phenotype, namely aberrant p53 expression, isocitrate dehydrogenase 1 mutation (R132H substitution) expression, mutant ATRX (αthalassemia/mental retardation syndrome X‐linked) expression and mutant DAXX (death‐domain‐associated protein) expression by immunohistochemistry (IHC). Overexpression of p16INK4A was examined immunohistologically in a subset of cases. Many of the tumours associated with death or recurrence demonstrated APBs commensurate with ALT telomere maintenance. However, all uterine STUMP (4/4), and vaginal STUMP (2/2) patients, and almost all LMS patients (88.4%, 23/26, including 90% (9/10) of stage 1 LMS cases), who had died of disease or who had recurrent disease, displayed loss of ATRX or DAXX expression. Loss of ATRX or DAXX expression identified poor prognosis (95% CI 2.1 to 40.8, p < 0.003), in the LMS group. Thus, loss of ATRX or DAXX expression in uterine smooth muscle tumours identifies a clinically aggressive molecular subtype of early stage LMS and when histopathological features are problematic such as in STUMP. As ATRX and DAXX IHC is readily performed in diagnostic laboratories these are potentially useful for routine histopathological classification and management.

Topoisomerase II inhibition suppresses the proliferation of telomerase-negative cancers.

Telomere maintenance is required for chromosome stability, and telomeres are typically elongated by telomerase following DNA replication. In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. Previous studies have indicated that yeast Sgs1 and Top3 may work together to remove highly negative supercoils that are generated from recombination. However, the mechanism by which cells eradicate highly positive supercoils during recombination remains unclear. In the present study, we demonstrate that TOP2 is involved in telomere-telomere recombination. Disturbance of telomeric structure by RIF1 or RIF2 deletion alleviates the requirement for TOP2 in telomere-telomere recombination. In human telomerase-negative alternative lengthening of telomere (ALT) cells, TOP2α or TOP2β knockdown decreases ALT-associated PML bodies, increases telomere dysfunction-induced foci and triggers telomere shortening. Similar results were observed when ALT cells were treated with ICRF-193, a TOP2 inhibitor. Importantly, ICRF-193 treatment blocks ALT-associated phenotypes in vitro, causes telomere shortening, and inhibits ALT cell proliferation inmice. Taken together, these findings imply that TOP2 is involved in the ALT pathway, perhaps by resolving the highly positive supercoil structure at the front of the helicase. Inhibition of topoisomerase II may be a promising therapeutic approach that can be used to prevent cell proliferation in ALT-type cancer cells.

WRN Loss Induces Switching of Telomerase-Independent Mechanisms of Telomere Elongation

Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.

Telomerase Suppresses Formation of ALT-Associated Single-Stranded Telomeric C-Circles

Telomere maintenance is an essential characteristic of cancer cells, most commonly achieved by activation of telomerase. Telomeres can also be maintained by a recombination-based mechanism, alternative lengthening of telomeres (ALT). Cells using ALT are characterized by the presence of ALT-associated promyelocytic leukemia (PML) bodies (APB), long, heterogeneously sized telomeres, extrachromosomal telomeric circular DNA, and elevated telomeric recombination. Consistent with other reports, we found that liposarcomas containing APBs, but lacking telomerase expression, always contained C-rich circles (C-circles), and these C-circles were never present in the absence of APBs, indicating a tight link between these features in ALT cells. However, a rare subgroup of tumors showing evidence of telomere maintenance by both telomerase and ALT did not contain C-circles. To test the hypothesis that telomerase expression disrupts the tight link between APBs and C-circles, we used ALT cell lines that were engineered to express telomerase. Introduction of telomerase activity in these ALT cells resulted in, on average, shorter telomeres with retention of APBs. However, at high passage, the level of C-circles was significantly reduced, which was paralleled by a switch from C-strand overhangs to G-strand overhangs. We propose that by extending critically short telomeres in these cells, telomerase is disrupting a key step in the ALT pathway necessary for production and/or maintenance of C-circles.

The F-box protein β-TrCP promotes ubiquitination of TRF1 and regulates the ALT-associated PML bodies formation in U2OS cells

The telomeric repeat binding protein 1 (TRF1) is a major factor of the mammalian telosome/shelterin and negatively regulates telomere length by inhibiting access of telomerase at telomere termini in telomerase-positive cells. In telomerase-negative cancer cells, TRF1 also plays a critical role in the mechanism called alternative lengthening of telomeres (ALT) and is essential for formation of the ALT-associated PML bodies (APBs). It was reported that TRF1 can be degraded by the ubiquitin–proteasome pathway, involving in two regulation factors, Fbx4 and RLIM. Here, we reported that β-TrCP1, a member of the F-box family protein with ubiquitin ligase activity, is a novel TRF1-associating protein. β-TrCP1 interacts with TRF1 in vivo and in vitro and promotes its ubiquitination. Overexpression of β-TrCP1 reduces endogenous TRF1 protein levels, while inhibition of β-TrCP1 by siRNA stabilizes TRF1. Moreover, we found that β-TrCP1 is essential for regulation of promyelocytic leukemia body recruitment of TRF1 in U2OS cells. These results reveal that β-TrCP1 is involved in the negative regulation of TRF1 and represents a new pathway for APB formation in telomerase-negative cells.

The telomere-associated homeobox-containing protein TAH1 participates in telomere maintenance in ALT Cells

The majority of cancer cells rely on elevated telomerase expression and activity for rapid growth and proliferation. Telomerase-negative cancer cells, by contrast, often employ the alternative lengthening of telomeres (ALT) pathway to maintain telomeres. ALT cells are characterized by long and dynamic telomeres and the presence of ALT-associated promyelocytic leukemia (PML) bodies (APBs). Previous work has shown the importance of APBs to the ALT pathway, but their formation and precise role remain unclear. Here, we demonstrate that a homeobox-containing protein known as HMBOX1 can directly bind telomeric double-stranded DNA and associate with PML nuclear bodies. Hence, we renamed this protein TAH1 for telomere-associated homeobox-containing protein 1. TAH1 knockdown significantly reduced the number of APBs and led to an increase in DNA damage response signals at telomeres. Importantly, TAH1 inhibition also notably reduced the presence of telomere C-circles, indicating altered ALT activity. Our findings point to TAH1 as a novel link between pathways that regulate DNA damage responses, PML nuclear bodies, and telomere homeostasis in ALT cells, and provide insight into how ALT cells may achieve sustained growth and proliferation independent of the telomerase.

Human Sarcomas Are Mosaic for Telomerase-Dependent and Telomerase-Independent Telomere Maintenance Mechanisms: Implications for Telomere-Based Therapies

Telomere shortening necessitates that tumor cells activate a telomere maintenance mechanism (TMM) to support immortalization. Although most tumor cells activate expression of the enzyme telomerase, some cells elongate telomeres in a telomerase-independent manner, termed alternative lengthening of telomeres (ALT). Previous studies have evaluated the presence of telomerase or ALT mechanisms or both in a variety of tumor types. Our studies also show that TMMs are not mutually exclusive in some tumors. In contrast, our IHC analyses of human sarcomas identified a subset of tumors with some cells containing ALT-associated PML bodies, a hallmark of ALT, and separate cells expressing telomerase in the same tumor. By using a second set of human osteosarcomas, we merged IHC and biochemical analyses to characterize more fully the tumor TMM. The IHC data reveal the presence of both telomerase- and ALT-positive tumor cells in samples that demonstrate characteristics of both telomerase and ALT in biochemical assays. These assays, which measure telomere length and telomerase activity of tumor extracts, are conventionally used to classify tumor TMM. Our results suggest that TMM is not a single or perhaps static characteristic of some tumors and that TMM heterogeneity should be considered in tumor stratification. Furthermore, clinical interest in telomere-based therapies may necessitate accurate characterization of tumor TMM before treatment to maximize therapeutic efficacy.

A new level of protection

This study identifies a new way to maintain telomere integrity involving nucleostemin. Loss of nucleostemin increased the formation of telomere damage foci, and in telomerase-inactive cells (which instead use alternative lengthening of telomeres (ALT)), it decreased the number of ALT-associated PML bodies (APBs; which may have a role in damage repair by homologous recombination (HR)) and the percentage of telomeres associated with APBs. So how does nucleostemin protect telomeres? In ALT cells, nucleostemin increased the sumoylation of the telomere protein TRF1 (which is required for APB formation) and promoted its association with the PML body protein PML-IV. Furthermore, the sumoylated TRF1–PML-IV interaction was induced following DNA damage in a nucleostemin-dependent manner, and this was required for the recruitment of the HR protein RAD51 to damage sites. Importantly, this mechanism was conserved in telomerase-active cells.

Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1

Continuously dividing cells must be protected from telomeric and nontelomeric DNA damage in order to maintain their proliferative potential. Here, we report a novel telomere-protecting mechanism regulated by nucleostemin (NS). NS depletion increased the number of telomere damage foci in both telomerase-active (TA(+)) and alternative lengthening of telomere (ALT) cells and decreased the percentage of damaged telomeres associated with ALT-associated PML bodies (APB) and the number of APB in ALT cells. Mechanistically, NS could promote the recruitment of PML-IV to SUMOylated TRF1 in TA(+) and ALT cells. This event was stimulated by DNA damage. Supporting the importance of NS and PML-IV in telomere protection, we demonstrate that loss of NS or PML-IV increased the frequency of telomere damage and aberration, reduced telomeric length, and perturbed the TRF2(ΔBΔM)-induced telomeric recruitment of RAD51. Conversely, overexpression of either NS or PML-IV protected ALT and TA(+) cells from telomere damage. This work reveals a novel mechanism in telomere protection.

Telomere maintenance mechanisms in malignant peripheral nerve sheath tumors: expression and prognostic relevance

This study investigated the prevalence and the prognostic relevance of the 2 known telomere maintenance mechanisms (TMMs), telomerase activity (TA) and alternative lengthening of telomeres (ALT), in malignant peripheral nerve sheath tumors (MPNST). In 57 specimens from 49 patients with MPNST (35 sporadic, 14 neurofibromatosis type 1-related), TA was determined using the telomeric repeat amplification protocol, and ALT was detected by assaying ALT-associated promyelocytic leukemia bodies (APB) and terminal restriction fragment (TRF) length distribution. TA or ALT (defined on the basis of APB) alone was found in 24.6% or 26.3% of the lesions, respectively, whereas 6 cases (10.5%) were TA+/ALT+. A concordance between APB and TRF results in defining the ALT status was observed in 44 of 57 cases (77.2%; P < .0001). TA was more frequently expressed in samples from patients with neurofibromatosis type 1 than in those with sporadic disease (60% vs 29.4%, P = 0.087). In the overall series, TA proved to be prognostic for 5-year disease-specific death (hazard ratio, 3.78; 95% confidence interval [CI], 1.60-8.95; P = .002), even when adjusted for the presence of neurofibromatosis type 1 (hazard ratio, 4.22; 95% CI, 1.804-9.874; P = .001) and margin status after surgery (hazard ratio, 5.78; 95% CI, 2.19-15.26; P < .001). Conversely, ALT did not significantly affect clinical outcome of MPNST using either APB expression (hazard ratio, 1.25; 95% CI 0.54-2.89; P = 0.605) or TRF distribution (hazard ratio, 0.57; 95% CI, 0.17-1.96; P = .375) as the detection approach. Our results indicate for the first time that both TMMs, TA and ALT, are present in MPNST and differentially affect patient prognosis.

NIP45 Promotes Telomere Targeting to PML Bodies in ALT Cells

Telomere length maintenance is critical for continued cell proliferation. Certain cancer cells do not have active telomerase and use homologous recombination to lengthen their telomeres, a mechanism referred to as alternative lengthening of telomeres (ALT). We have previously shown that the SMC5/6 complex is required for telomere length maintenance in ALT cells. The SMC5/6 component MMS21 sumoylates multiple telomere‐binding proteins and promotes telomere targeting to ALT‐associated PML bodies (APBs). In this study, we show that the SUMO‐like protein NIP45 is also required for targeting telomeres to APBs. NIP45 itself localizes to PML bodies and promotes MMS21‐dependent sumoylation of telomere‐binding proteins. The C‐terminal SUMO‐like domain of NIP45 is sufficient to restore both APB formation and sumoylation of telomere‐binding proteins in NIP45 RNAi cells. Our results suggest that NIP45 enables PML‐body recruitment and sumoylation of telomere‐binding proteins, thus promoting APB formation in ALT cells.This work was supported by a National Research Service Award grant 5 T32 GM 8203‐24 to Demetra D. Farley.

Prognostic Value of Alternative Lengthening of Telomeres–Associated Biomarkers in Uterine Sarcoma and Uterine Carcinosarcoma

A subset of cancer cells maintains telomere lengths in a telomerase-independent manner known as the alternative lengthening of telomeres (ALT). The goal of this study was to evaluate the frequency of ALT in uterine sarcoma and carcinosarcoma and to assess its association with clinical parameters. Retrospectively collected paraffin blocks from 41 patients with uterine sarcomas and carcinosarcomas were analyzed for ALT-associated promyelocytic leukemia bodies (APBs), which are a significant feature of ALT cells, using combined immunofluorescence and telomere fluorescence in situ hybridization. In addition, a C-circle assay and human telomerase reverse transcriptase immunohistochemistry were performed to support these results. The APB assay and C-circle assay indicated that 46.3% (19/41 cases) and 36.4% (8/22 cases) of sarcomas of the uterus, respectively, were positive for ALT. Alternative lengthening of telomerase positivity was correlated with high-grade uterine sarcoma and parameters indicative of an aggressive tumor, such as tumor size (P = 0.033) and mitotic index (P = 0.001); ALT positivity was negatively correlated with human telomerase reverse transcriptase reactivity (P = 0.036). In a survival analysis, the presence of APBs was found to be a poor prognostic factor for disease-free survival (P = 0.018) and overall survival (P = 0.021). Alternative lengthening of telomeres is a prevalent mechanism in uterine sarcomas and carcinosarcomas and is associated with the aggressiveness of the tumor and tumor progression. Importantly, ALT positivity is an indicator of poor prognosis for patients with uterine sarcoma and carcinosarcoma.

The alternative lengthening of telomeres pathway may operate in non‐neoplastic human cells

The alternative lengthening of telomeres (ALT) mechanism represents an alternative to the enzyme telomerase in the maintenance of mammalian telomeres in 25-60% of sarcomas and a minority of carcinomas (about 5-15%). ALT-positive cells are distinguished by long and heterogeneous telomere length distributions by terminal restriction fragment (TRF) Southern blotting. Another diagnostic marker of ALT is discrete nuclear co-localized signals of telomeric DNA and the promyelocytic leukaemia protein (PML), referred to as ALT-associated PML bodies (APBs). Recently, we detected smaller sized co-localized PML and telomere DNA (APB-like) bodies in endothelial cells adjacent to astrocytoma tumour cells in situ. In this study, we examined a wide variety of non-neoplastic tissues, and report that co-localized signals of PML and telomere DNA are present in endothelial, stromal, and some epithelial cells. Co-localized signals of PML and telomere DNA showed an increased frequency in non-neoplastic cells with DNA damage. These results suggest that a mechanism similar to that in ALT-positive tumours also operates in non-neoplastic cells, which may be activated by DNA damage.

De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation

Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components. We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: (1) PML and Sp100 as the constituting components of PML nuclear bodies, (2) telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), (3) the DNA repair protein NBS1 and (4) the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.

Abstract B57: Coexistence of telomerase-dependent and -independent telomere maintenance mechanisms in human osteosarcoma tumors

Abstract Telomeres, the terminal-most structures of chromosomes, are critical to maintain the integrity of chromosomal DNA. As tumor cells bypass normal cell cycle controls and expand their proliferative capacity, accelerated telomere erosion necessitates the activation of a telomere maintenance mechanism. This mechanism can be telomerase-dependent or -independent. Telomerase-independent maintenance is termed alternative lengthening of telomeres, or ALT, and may represent an amalgamation of several recombination-associated pathways. ALT likely uses other telomeres or extrachromosomal telomeric DNA as a template for recombination-based addition of telomere repeats to a shortened chromosome. While most human tumors use telomerase to maintain telomere length, a subset uses ALT. Osteosarcomas are more commonly in this category, as almost 60% demonstrate ALT characteristics. Previous studies to analyze mechanisms of telomere maintenance have shown that while some tumors are ALT or telomerase-dependent, a small number demonstrates mixed Results: both a heterogeneous telomere length consistent with ALT and telomerase activity, incompatible with ALT, or vice versa (reviewed in Henson and Reddel, 2010). These results suggest the idea of a mixed population of cells within some tumors that uses both telomerase-dependent and -independent telomere maintenance mechanisms; however, this has not yet been confirmed. Our studies have used a human osteosarcoma tissue microarray to demonstrate both ALT and telomerase-dependent cells within the same tumor. Tumor sections were stained for PML, TRF2 and telomerase, followed by analysis with the Nuance Multispectral Imaging System. Some tumors (4/6) displayed robust staining and colocalization of PML and TRF2, indicative of ALT-associated PML bodies (APBs), a marker of ALT cells. Some of these tumors (3/4) also contained cells staining strongly for telomerase that were exclusive of ALT-specific colocalization. These two types of cells within this subset of tumors were distinct from one another. To confirm these initial findings, five frozen human osteosarcoma samples were evaluated to correlate in situ staining results with classical methods of gross telomere classification, characterization of telomere length via telomere restriction fragment (TRF) Southern blot and identification of telomerase activity via the telomere repeat amplification protocol (TRAP). Two tumors displayed ALT characteristics only, exhibiting heterogeneous telomere lengths with TRF Southern blots and negative findings with TRAP; one tumor displayed positive TRAP results and more homogeneous telomere lengths associated with telomerase activity. Of the remaining two tumors, one was characterized by telomerase-associated homogenous telomere length and one was characterized by telomerase-independent heterogeneous telomere length by TRF Southern blots; both tumor lysates displayed variable and weak telomerase activity with TRAP. These data suggest that cells within these tumors use more than one mechanism of telomere maintenance. Tumor sections from these five osteosarcomas are now being analyzed using our in situ immunohistochemistry methods. Given recent interest in telomerase-targeted cancer therapies, the coexistence of both telomere maintenance mechanisms within a single tumor would have significant implications for understanding the mechanisms of telomere maintenance and for developing precise therapies to target ALT mechanisms of telomere elongation. Henson, J. D. and Reddel, R. R. Assaying and investigating Alternative Lengthening of Telomeres activity in human cells and cancers. FEBS Lett., 584: 3800–3811, 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B57.

Alternative lengthening of telomeres—An enhanced chromosomal instability in aggressive non‐MYCN amplified and telomere elongated neuroblastomas

Telomere length alterations are known to cause genomic instability and influence clinical course in several tumor types, but have been little investigated in neuroblastoma (NB), one of the most common childhood tumors. In the present study, telomere-dependent chromosomal instability and telomere length were determined in six NB cell lines and fifty tumor biopsies. The alternative lengthening of telomeres (ALT) pathway was assayed by scoring ALT-associated promyelocytic leukemia (PML) bodies (APBs). We found a reduced probability of overall survival for tumors with increased telomere length compared to cases with reduced or unchanged telomere length. In non-MYCN amplified tumors, a reduced or unchanged telomere length was associated with 100% overall survival. Tumor cells with increased telomere length had an elevated frequency of APBs, consistent with activation of the ALT pathway. The vast majority of tumor biopsies and cell lines exhibited an elevated rate of anaphase bridges, suggesting telomere-dependent chromosomal instability. This was more pronounced in tumors with increased telomere length. In cell lines, there was a close correlation between lack of telomere-protective TTAGGG-repeats, anaphase bridging, and remodeling of oncogene sequences. Thus, telomere-dependent chromosomal instability is highly prevalent in NB, and may contribute to the complexity of genomic alterations as well as therapy resistance in the absence of MYCN amplification and in this tumor type.

Pilocytic Astrocytomas Have Telomere-Associated Promyelocytic Leukemia Bodies without Alternatively Lengthened Telomeres

Telomere maintenance by either telomerase activity or the recombination-mediated alternative lengthening of telomeres (ALT) mechanism is a hallmark of cancer. Tumors that use ALT as their telomere maintenance mechanism are characterized by long telomeres of great heterogeneity in length and by specific nuclear structures of co-localized promyelocytic leukemia protein and telomere DNA, called ALT-associated promyelocytic leukemia bodies (APBs). Recent advances have revealed a direct role for APBs in telomere recombination in ALT-positive cells. In this study, we investigated the possibility that APBs could occur before the long 'alternatively' lengthened telomeres arise, particularly in low-grade tumors. We measured APBs, telomere length, and telomerase activity in 64 astrocytomas inclusive of grade 1-4 tumors. Almost all grade 1-3 tumors (93%) were APB-positive using published criteria. Grade 2-3 APB-positive tumors also had long telomeres and were confirmed as ALT positive. However, grade 1 tumors lacked long telomeres and were therefore classified as ALT negative, but positive for telomere-associated promyelocytic leukemia bodies (TPB). This is the first report of a TPB-positive but ALT-negative tumor, and suggests that low-grade tumors have the foundation for recombinational telomere repair, as in ALT. Further work is warranted to characterize the TPB-positive phenotype in other early malignancies, as well as to determine whether TPBs predispose to telomere maintenance by ALT.

P53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML-NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML-NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53-dependent growth inhibition. ALT-associated PML bodies (APBs) are specialized PML-NBs that include telomere-repeat binding-factor TRF1 and are exclusively in telomerase-negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell-cycle and p53 responses in ALT-positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin-3a. Both IR and Nutlin-3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin-3a. p53 was recruited to PML-NBs 3-4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML-NBs marked sites of apparently unrepaired DNA double-strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin-3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin-3a-treated cells. These data indicate (1) p53 is recruited to PML-NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53-p21 pathway activation increases the percentage of APB-positive cells, (3) p21 and p53 are recruited to ALT-associated PML-NBs after Nutlin-3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance.