Search

BackgroundAltered nuclei are ubiquitous in cancer, with changes in nuclear size, shape, and coloration accompanying cancer progression. However, comprehensive quantification of nuclear morphology across a whole-slide histologic image remains a challenge. Here, we describe the development of a pan-tissue, deep learning-based digital pathology pipeline for exhaustive nucleus detection, segmentation, and classification and the utility of this pipeline for nuclear morphologic biomarker discovery.MethodsManually-collected nucleus annotations (>29,000) were used to train an object detection and segmentation model for identifying nuclei, which was deployed to segment nuclei in H&E-stained slides from the BRCA, LUAD, and PRAD TCGA cohorts. Interpretable features describing the shape, size, color, and texture of each nucleus were extracted from model predictions. These quantitative nuclear features were compared to available metrics, including measurements of genomic instability, gene expression, and prognosis.ResultsThe nuclear segmentation and classification model trained herein performed comparably to previously reported models. Features extracted from the model revealed differences sufficient to distinguish between BRCA, LUAD, and PRAD. Furthermore, cancer cell nuclear area was associated with increased genomic instability, as measured by aneuploidy score and homologous recombination deficiency. In BRCA, increased fibroblast nuclear area was indicative of poor progression-free and overall survival and was associated with gene expression signatures related to extracellular matrix remodeling and anti-tumor immunity.ConclusionsWe developed a powerful pan-tissue approach for nucleus segmentation and featurization, enabling the construction of predictive models and the identification of features linking nuclear morphology with clinically-relevant prognostic biomarkers across multiple cancer types.

Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. Here, a robust chromogenicin situhybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). This new assay (“Telo-CISH”) produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.

Table S1. Sequences of oligonucleotides for CRISPR, telomere assays, and RT-qPCR. Table S2. Mean telomere lengths assessed from TRF southern blots of LAPC-4 EV and ATRXKO clones, and CWR22Rv1 EV and ATRXKO clones. Table S3. Microarray results of top 10 differentially expressed genes ranked by FDR adjusted P-value, comparing parental ATRX WT cell lines (LAPC-4 and CWR22Rv1) versus ATRXKO cell lines (LAPC-4 ATRXKO1-3 and CWR22Rv1 ATRXKO1-3). Table S4. Microarray results of top 10 differentially expressed genes with FDR adjusted P-value < 0.005 ranked by absolute fold-change, comparing LAPC-4 ATRXKO1-3 clones versus CWR22Rv1 ATRXKO1-3 clones.

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.

Figure S1. LAPC-4 and CWR22RV1 are telomerase-positive ALT-negative cell lines. Figure S2. ATRXKOs were generated in LAPC-4 and CWR22Rv1 cells. Figure S3. Representative telomere restriction fragment (TRF) southern blots and signal intensity traces. Figure S4. TERT and TERC expression levels. Figure S5. CRISPR cas9 genome editing strategies targeting the TERC locus. Figure S6. RT-qPCR validation of genes differentially expressed in microarray.

Supplementary Materials and Methods

Supplemental Figure 1. (A) ALT-positive PanNET patients with lymph node metastasis (median survival, 131 months) had a tendency of better survival than ALT-negative PanNET patients with nodal metastasis (54 months, HR=0.31, 95% CI=0.08-1.19; p=0.088). (B) ALT-positive PanNET patients with distant metastasis (overall 5-year survival rate, 75.0%) trended toward better survival than ALT-negative patients (25.0%), but was not statistically significant (p=0.118).

Purpose: Alternative lengthening of telomeres (ALT), a telomerase-independent telomere maintenance mechanism, is strongly associated with ATRX and DAXX alterations and occurs frequently in pancreatic neuroendocrine tumors (PanNET).Experimental Design: In a Korean cohort of 269 surgically resected primary PanNETs and 19 sporadic microadenomas, ALT status and nuclear ATRX and DAXX protein expression were assessed and compared with clinicopathologic factors.Results: In PanNETs, ALT or loss of ATRX/DAXX nuclear expression was observed in 20.8% and 19.3%, respectively, whereas microadenomas were not altered. ALT-positive PanNETs displayed a significantly higher grade, size, and pT classification (all, P < 0.001). ALT also strongly correlated with lymphovascular (P < 0.001) and perineural invasion (P = 0.001) and the presence of lymph node (P < 0.001) and distant metastases (P = 0.002). Furthermore, patients with ALT-positive primary PanNETs had a shorter recurrence-free survival [HR = 3.38; 95% confidence interval (CI), 1.83–6.27; P < 0.001]. Interestingly, when limiting to patients with distant metastases, those with ALT-positive primary tumors had significantly better overall survival (HR = 0.23; 95% CI, 0.08–0.68; P = 0.008). Similarly, tumors with loss of ATRX/DAXX expression were significantly associated with ALT (P < 0.001), aggressive clinical behavior, and reduced recurrence-free survival (P < 0.001). However, similar to ALT, when limiting to patients with distant metastases, loss of ATRX/DAXX expression was associated with better overall survival (P = 0.003).Conclusions: Both primary ALT-positive and ATRX/DAXX-negative PanNETs are independently associated with aggressive clinicopathologic behavior and displayed reduced recurrence-free survival. In contrast, ALT activation and loss of ATRX/DAXX are both associated with better overall survival in patients with metastases. Therefore, these biomarkers may be used as prognostic markers depending on the context of the disease. Clin Cancer Res; 23(6); 1598–606. ©2016 AACR.

Correlation between ALT status and ATRX and DAXX protein expression.

(A) ALT-positive PanNET patients with lymph node metastasis (median survival, 131 months) had a tendency of better survival than ALT-negative PanNET patients with nodal metastasis (54 months, HR=0.31, 95% CI=0.08-1.19; p=0.088). (B) ALT-positive PanNET patients with distant metastasis (overall 5-year survival rate, 75.0%) trended toward better survival than ALT-negative patients (25.0%), but was not statistically significant (p=0.118).

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM-giant cell GBM-has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM.

Acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MAcNECs) of the pancreas are extremely rare carcinomas with a significant component with acinar differentiation. To date, the clinicopathological behaviours of these neoplasms remain unclear. In this study, we evaluated the histopathological and molecular characteristics of 20 ACCs and 13 MAcNECs and compared them to a cohort of 269 well-differentiated pancreatic neuroendocrine tumours (PanNETs). Compared to PanNETs, both ACCs and MAcNECs had an advanced pT classification (p

Telomeres, the repetitive DNA elements at chromosome ends, are pivotal for maintenance of genome integrity. Previous studies from our group and others have highlighted the translational potential of tissue-based telomere length measurements to address the clinical challenge of improving diagnosis, individualized risk stratification, and accurate prognostication of different diseases. Here, we describe a high-throughput method that quantitates cell type-specific telomere lengths at a single cell level in archival tissues from patient cohorts for research on prognosis. This approach is based on telomere-specific fluorescence in situ hybridization (FISH) combined with multiplex immunostaining for cell type-specific antibodies, followed by semi-automated slide scanning and multi-channel acquisition of fluorescent images using the TissueFAXS Plus microscopy workstation and TissueQuest software (TissueGnostics). Here, we demonstrate that this method is sufficiently robust and reproducible to detect biologically significant differences in telomere lengths in archived tissues either on whole slides or sampled across tissue microarrays, which is essential when assessing prognosis in large patient cohorts.

High-grade glioma, including anaplastic astrocytoma and glioblastoma (GBM) patients, have a poor prognosis due to the lack of effective treatments. Therefore, the development of new therapeutic strategies to treat these gliomas is urgently required. Given that high-grade gliomas frequently harbor mutations in the SNF2 family chromatin remodeler ATRX, we performed a screen to identify FDA-approved drugs that are toxic to ATRX-deficient cells. Our findings reveal that multi-targeted receptor tyrosine kinase (RTK) and platelet-derived growth factor receptor (PDGFR) inhibitors cause higher cellular toxicity in high-grade glioma ATRX-deficient cells. Furthermore, we demonstrate that a combinatorial treatment of RTKi with temozolomide (TMZ)–the current stand-ard of care treatment for GBM patients–causes pronounced toxicity in ATRX-deficient high-grade glioma cells. Our findings suggest that combinatorial treatments with TMZ and RTKi may increase the therapeutic window of opportunity in patients who suffer high-grade gliomas with ATRX mu-tations. Thus, we recommend incorporating the ATRX status into the analyses of clinical trials with RTKi and PDGFRi., This work was supported by grants from Danish National Research Foundation (DNRF115), Danish Cancer Society (KBVU-2017_R167-A11063), European Research Council (ERC-2015-STG- 679068), Nordea-fonden (02-2017-1749) and the Spanish Ministry of Science and Innovation (PID2020- 119329RB-I00). David Pladevall-Morera was supported with a PhD scholarship from the Lundbeck Foundation (R218-2016-415) and funding from Dansk Kræftforskningsfond. María Castejón-Griñán holds an Incorporación fellowship from the Junta de Andalucía. Paula Aguilera was supported with a Juan de la Cierva formación fellowship from the MICINN and an Incorporación fellowship from the Junta de Andalucía. Toyota Fonden and Læge Sofus Carl Emil Friis og hustru Olga Doris Fonden funded the acquisition of the high-content microscope used in this study.

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.

Neurofibromatosis type-1 is a familial genetic syndrome associated with a predisposition to develop peripheral and central nervous system neoplasms. We have previously reported on a subset of gliomas developing in these patients with morphologic features resembling subependymal giant cell astrocytoma, but the molecular features of these tumors remain undefined. A total of 14 tumors were studied and all available slides were reviewed. Immunohistochemical stains and telomere-specific FISH were performed on all cases. In addition, next-generation sequencing was performed on 11 cases using a platform targeting 644 cancer-related genes. The average age at diagnosis was 28 years (range: 4–60, 9F/5M). All tumors involved the supratentorial compartment. Tumors were predominantly low grade (n = 12), with two high-grade tumors, and displayed consistent expression of glial markers. Next-generation sequencing demonstrated inactivating NF1 mutations in 10 (of 11) cases. Concurrent TSC2 and RPTOR mutations were present in two cases (1 sporadic and 1 neurofibromatosis type-1-associated). Interestingly, alternative lengthening of telomeres was present in 4 (of 14) (29%) cases. However, an ATRX mutation associated with aberrant nuclear ATRX expression was identified in only one (of four) cases with alternative lenghtening of telomeres. Gene variants in the DNA helicase RECQL4 (n = 2) and components of the Fanconi anemia complementation group (FANCD2, FANCF, FANCG) (n = 1) were identified in two alternative lenghtening of telomere-positive/ATRX-intact cases. Other variants involved genes related to NOTCH signaling, DNA maintenance/repair pathways, and epigenetic modulators. There were no mutations identified in DAXX, PTEN, PIK3C genes, TP53, H3F3A, HIST1H3B, or in canonical hotspots of IDH1, IDH2, or BRAF. A subset of subependymal giant cell astrocytoma-like astrocytomas are alternative lenghtening of telomere-positive and occur in the absence of ATRX alterations, thereby suggesting mutations in other DNA repair/maintenance genes may also facilitate alternative lenghtening of telomeres. These findings suggest that subependymal giant cell astrocytoma-like astrocytoma represents a biologically distinct group that merits further investigation.

ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and IDH-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We found that ATRX binds regulatory elements of cell cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intra-cranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wildtype controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display epigenetic dysregulation of cell cycle phase transitions, which opens a new window for therapies targeting this unique phenotype.

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown.DesignAn international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS).ResultsATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (pConclusionsATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM-giant cell GBM-has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM.

Background In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. Methods Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. Results In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Conclusions Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded byTERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date ofTERTpromoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the coreTERTpromoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250TTERTpromoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observedTERTexpression in cancer cells. Our findings suggest that hypermethylation of theTERTpromoter alleles signals transcriptional repression of those alleles, leading to the attenuation ofTERTactivation in cancer cells.SIGNIFICANCEHypermethylation of theTERTpromoter alleles to attenuateTERTactivation in cancer cells may account for the modest activation ofTERTexpression in most cancers.

Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fifty-seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3–75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomere-specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3-K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRX- (20/24, 83%) or ALT-/ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3-K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PA-A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3-K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTpWT-IDHWT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTpWT-IDHWT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDHWT-TERTSV), and an ALT-positive subgroup (IDHWT-ALT) with mutations in ATRX or SMARCAL1., Glioblastoma can be classified based on IDH and TERT promoter mutations, but ~20% of glioblastoma do not have these mutations (TERTpWT-IDHWT glioblastoma). Here, the authors present a genetic landscape of TERTpWT-IDHWT glioblastoma, identifying a telomerase-positive subgroup driven by TERT-structural rearrangements and an ALT-positive subgroup with mutations in ATRX or SMARCAL1.

The presence of Alternative lengthening of telomeres (ALT) and/or ATRX loss, as well as the role of other telomere abnormalities, have not been formally studied across the spectrum of NF1-associated solid tumors. Utilizing a telomere-specific FISH assay, we classified tumors as either ALT-positive or having long (without ALT), short, or normal telomere lengths. A total of 426 tumors from 256 NF1 patients were evaluated, as well as 99 MPNST tumor samples that were sporadic or of unknown NF1 status. In the NF1-glioma dataset, ALT was present in the majority of high-grade gliomas: 14 (of 23; 60%) in contrast to only 9 (of 47; 19%) low-grade gliomas (p = 0.0009). In the subset of ALT-negative glioma cases, telomere lengths were estimated and we observed 17 (57%) cases with normal, 12 (40%) cases with abnormally long, and only 1 (3%) case with short telomeres. In the NF1-associated malignant nerve sheath tumor (NF1-MPNST) set (n = 75), ALT was present in 9 (12%). In the subset of ALT-negative NF1-MPNST cases, telomeres were short in 9 (38%), normal in 14 (58%) and long in 1 (3%). In the glioma set, overall survival was significantly decreased for patients with ALT-positive tumors (p

Patients with glioma whose tumors carry a mutation in isocitrate dehydrogenase 1 (IDH1 R132H ) are younger at diagnosis and live longer. IDH1 mutations co-occur with other molecular lesions, such as 1p/19q codeletion, inactivating mutations in the tumor suppressor protein 53 (TP53 ) gene, and loss-of-function mutations in alpha thalassemia/mental retardation syndrome X-linked gene ( ATRX ). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1 R132H mutation. The current molecular classification of LGGs is based, partly, on the distribution of these mutations. We developed a genetically engineered mouse model harboring IDH1 R132H , TP53 and ATRX inactivating mutations, and activated NRAS G12V. Previously, we established that ATRX deficiency, in the context of wild-type IDH1, induces genomic instability, impairs nonhomologous end-joining DNA repair, and increases sensitivity to DNA-damaging therapies. In this study, using our mouse model and primary patient-derived glioma cultures with IDH1 mutations, we investigated the function of IDH1 R132H in the context of TP53 and ATRX loss. We discovered that IDH1 R132H expression in the genetic context of ATRX and TP53 gene inactivation (i) increases median survival in the absence of treatment, (ii) enhances DNA damage response (DDR) via epigenetic up-regulation of the ataxia-telangiectasia–mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinases 1 and 2, essential kinases in the DDR, restored the tumors’ radiosensitivity. Translation of these findings to patients with IDH1 132H glioma harboring TP53 and ATRX loss could improve the therapeutic efficacy of radiotherapy and, consequently, patient survival.

In cancers, the epigenetic regulation of telomerase expression is not fully characterized, with multiple conflicting reports. In particular, the role of DNA methylation of cytosine residues in the control of TERT expression remains unclear. Here, we have carried out the most comprehensive characterization of TERT promoter methylation using ultra-deep bisulfite sequencing spanning more than 310 CpGs within and surrounding the core TERT promoter in 96 different cell lines, including primary, immortalized, and cancer cell types, as well as in control and reference samples. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent G228A and G250A TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. Notably, in cancers, the hypermethylated alleles were associated with repressed expression while remaining unmethylated alleles were enriched with the open chromatin marks, H3K4me3 and H3K27ac. Furthermore, the hypomethylated alleles of the TERT promoter were responsible for the observed TERT expression in cancer. This association of hypomethylated alleles with active expression and hypermethylated alleles with repressed expression is particularly evident in cancers harboring TERT promoter mutations, and cancers displaying allele-specific expression. Reporter assays confirmed that DNA methylation of the TERT promoter greatly suppressed reporter expression in heterologous TERT promoter-reporter constructs. Overall, our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells. Citation Format: Mindy K. Graham, David Esopi, Jacqueline A. Brosnan-Cashman, Anuj Gupta, Angelo De Marzo, William G. Nelson, Christopher M. Heaphy, Alan K. Meeker, Sarah J. Wheelan, Srinivasan Yegnasubramanian. Pervasive promoter hypermethylation of silenced TERT alleles in human cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 157.

Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human cancer.

Gliomas are a leading cause of cancer mortality in children and adults and new targeted therapies are desperately needed. ATRX is a chromatin remodeling protein that is recurrently mutated in H3F3A-mutant pediatric GBM and IDH-mutant grade 2/3 adult glioma. We previously showed that loss of ATRX in glioma results in tumor growth and additional tumor mutations. However, the mechanism driving these phenotypes has not been fully established. We found that in ChIP-Seq datasets of mouse neuronal precursor cells (NPCs) and experimental models of human glioma cells, ATRX binds and regulates the chromatin state of promoters and enhancers for gene sets associated with regulation of the cell cycle G2/M checkpoint. In line with this, analysis of single-cell seq (sc-seq) data from IDH-mutant gliomas (n=16) shows that ATRX-mutant tumors (IDH-A) demonstrate a population of cycling cells with dysregulated cell cycle phase gene set expression when compared to ATRX-wildtype tumors (IDH-O). In glioma models, ATRX-deficient cells exhibit a seven-fold increase in mitotic index at 16 hours after sub-lethal radiation and enhanced activation of the master cell cycle regulator ATM with radiation. Treatment of ATRX-deficient gliomas with ATM inhibitors results in a selective increase in dysfunctional cell cycling and increased radio-sensitization in ATRX-deficient glioma cells. Using an ATM-luciferase reporter in orthotopically-implanted human GBM cells, both AZD0156 and AZD1390 demonstrate in vivo pathway inhibition. Mice intra-cranially implanted with ATRX-deficient GBM cells demonstrate a doubling of median survival compared to radiated controls (p=0.0018) when treated with AZD0156 combined with radiation. This study demonstrates that ATRX-deficient glioma display epigenetic dysregulation of the G2/M checkpoint, which opens a new window for therapies targeting this unique phenotype.

ATRX is a histone chaperone protein recurrently mutated in pediatric GBM. We previously confirmed its role in tumor progression and mutational burden in glioma. However, the mechanism which mediates the proliferative advantage of ATRX loss in pediatric GBM remains unexplained. Recent data revealed a distinct pattern of DNA binding sites of the ATRX protein using ChIP-seq in mouse neuronal precursor cells (mNPCs). Using the ATRX peaks identified in p53(-/-) mNPCs, we confirmed that ATRX binding sites were significantly enriched in gene promoters (p

Genes encoding proteins that regulate chromatin structure and DNA modifications [i.e., chromatin regulatory factors (CRF)] and genes encoding histone proteins harbor recurrent mutations in most human cancers. These mutations lead to modifications in tumor chromatin and DNA structure and an altered epigenetic state that contribute to tumorigenesis. Mutated CRFs have now been identified in most types of cancer and are increasingly regarded as novel therapeutic targets. In this review, we discuss DNA alterations in CRFs and how these influence tumor chromatin structure and function, which in turn leads to tumorigenesis. We also discuss the clinical implications and review concepts of targeted treatments for these mutations. Continued research on CRF mutations will be critical for our future understanding of cancer biology and the development and implementation of novel cancer therapies. Cancer Res; 77(2); 227–33. ©2017 AACR.

Purpose: Alternative lengthening of telomeres (ALT), a telomerase-independent telomere maintenance mechanism, is strongly associated with ATRX and DAXX alterations and occurs frequently in pancreatic neuroendocrine tumors (PanNET).Experimental Design: In a Korean cohort of 269 surgically resected primary PanNETs and 19 sporadic microadenomas, ALT status and nuclear ATRX and DAXX protein expression were assessed and compared with clinicopathologic factors.Results: In PanNETs, ALT or loss of ATRX/DAXX nuclear expression was observed in 20.8% and 19.3%, respectively, whereas microadenomas were not altered. ALT-positive PanNETs displayed a significantly higher grade, size, and pT classification (all, P < 0.001). ALT also strongly correlated with lymphovascular (P < 0.001) and perineural invasion (P = 0.001) and the presence of lymph node (P < 0.001) and distant metastases (P = 0.002). Furthermore, patients with ALT-positive primary PanNETs had a shorter recurrence-free survival [HR = 3.38; 95% confidence interval (CI), 1.83–6.27; P < 0.001]. Interestingly, when limiting to patients with distant metastases, those with ALT-positive primary tumors had significantly better overall survival (HR = 0.23; 95% CI, 0.08–0.68; P = 0.008). Similarly, tumors with loss of ATRX/DAXX expression were significantly associated with ALT (P < 0.001), aggressive clinical behavior, and reduced recurrence-free survival (P < 0.001). However, similar to ALT, when limiting to patients with distant metastases, loss of ATRX/DAXX expression was associated with better overall survival (P = 0.003).Conclusions: Both primary ALT-positive and ATRX/DAXX-negative PanNETs are independently associated with aggressive clinicopathologic behavior and displayed reduced recurrence-free survival. In contrast, ALT activation and loss of ATRX/DAXX are both associated with better overall survival in patients with metastases. Therefore, these biomarkers may be used as prognostic markers depending on the context of the disease. Clin Cancer Res; 23(6); 1598–606. ©2016 AACR.

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and show that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs non-homologous end joining (NHEJ) and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated, but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Telomeres consist of many kilobases of repeated TTAGGG sequences at the ends of chromosomes, protected by a sequence-specific protein cap. Telomeres progressively shorten with each cell division and ultimately become critically short; due to their extensive proliferation, cancer cells must find a way to counteract this telomere loss. While most cancers utilize telomerase to maintain their telomere length, about 5% of cancers use a telomerase-independent telomere maintenance strategy, termed alternative lengthening of telomeres (ALT). While, overall, ALT is rare in cancer (~5-10% of cases), this telomere maintenance mechanism is enriched in pediatric high-grade glioma (pHGG). Previous work in our laboratory suggests that nearly half of pHGG utilize ALT. To date, therapeutic options are largely ineffective for children with HGG, reflected in the five-year survival rate, which is less than 33%. Our goal is to better harness ALT as a clinical marker in pHGG, specifically by the identification of drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pHGG cell lines. Two of these six pHGG cell lines displayed features of ALT, including the presence of ALT-associated PML bodies and extrachromosomal telomeric DNA in the form of c-circles. Furthermore, these lines lacked measurable telomerase activity. It is well established that ATRX is commonly mutated in ALT-positive cancers, including pHGG. Interestingly, only one of these two ALT-positive pHGG cell lines displayed total loss of ATRX; the second cell line has an in-frame deletion in the ATRX gene, which may provide insight into the mechanism of how ATRX acts to suppress ALT. In addition, we have generated ATRX knockout cell lines from the four ALT-negative pHGG cells identified in this panel. Despite the strong link between ATRX loss and ALT in clinical samples, only one cell line displayed ALT characteristics after ATRX knockout. Comparison of the ALT-competent cell line to the ALT-resistant cell lines will yield important information about additional genetic or epigenetic events that allow ALT to occur. In conclusion, we have established in vitro models of ALT in pHGG cell lines based on endogenous ALT positivity and induction of ALT-like features based on ATRX modulation. These models will be invaluable resources as we strive to understand the molecular characteristics of ALT and translate these findings to better therapies for children with pHGG. Citation Format: Jacqueline A. Brosnan-Cashman, Mindy K. Graham, Anthony J. Rizzo, Kaylar Myers, Rebecca Zhang, Ezgi Göger, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Establishment and characterization of in vitro models of alternative lengthening of telomeres (ALT) in pediatric high-grade glioma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B14.

Telomeres consist of many kilobases of repeated TTAGGG sequences at the ends of chromosomes, protected by a sequence-specific protein cap. Telomeres shorten with each cell division and ultimately become critically short; due to their extensive proliferation, cancer cells must find a way to offset this telomere loss. Several cancer subtypes types, including neuroblastomas and sarcomas, use a telomerase-independent strategy for telomere maintenance, alternative lengthening of telomeres (ALT). The molecular event(s) through which ALT occurs in cancer remain poorly understood. Increased knowledge of these mechanism(s) is critical to our ability to effectively treat ALT(+) cancers. ALT(+) cancers harbor several hallmarks, including telomere DNA that manifests as unique, ultrabright foci by telomere-specific in situ hybridization (FISH) in histologic cancer specimens. Despite the fact that ALT-associated telomere DNA foci (ATDFs) are a reliable marker for ALT occurring in a cancer, not all cells within the cancer display them. Still, ATDFs are predicted to be the nodes for a recombination-based telomere elongation process in ALT, so may be critical for the ALT process. Despite their potential importance to the biology of ALT(+) cancers, cells containing ATDFs have not been specifically isolated and characterized. In order to study the ATDF biomarker, we have performed telomere-specific FISH on ALT(+) cancer cells in suspension, followed by isolation of the ATDF(+) cells by flow cytometry. Our approach is modified from a well-validated clinical assay (“Flow FISH”) used for measuring telomere lengths in blood. Ours is the first known application of Flow FISH to solid tumor cells. We have successfully and reproducibly isolated pure populations of ATDF(+) and ATDF(-) cells from two well-characterized ALT(+) cancer cell lines: SAOS2 (an osteosarcoma cell line) and SK-N-FI (a neuroblastoma cell line). Thus, we have overcome the technical barriers that are necessary to study ATDF(+) cancer cells in order to understand this putative biomarker and its mechanistic role in ALT-mediated telomere maintenance. Work is ongoing to study ATDF(+) versus ATDF(-) ALT-positive cancer cells through gene-expression profiling and proteomic analysis to 1) determine the existence of actionable pathways that will allow for therapeutic targeting of ATDF(+) cells, and 2) examine the mechanistic link of ATDFs to the ALT process. Therefore, for the first time, we have developed an approach to effectively and specifically study the population of cancer cells containing an ALT-specific biomarker. Through this ongoing work on multiple cancer types, we will unlock critical information about the global ALT mechanism, which will provide novel, actionable targets for ALT(+) cancers. Citation Format: Jacqueline A. Brosnan-Cashman, Christopher M. Heaphy, Alan K. Meeker. Isolation and characterization of cancer cells containing ultrabright telomere DNA foci associated with alternative lengthening of telomeres (ALT): A novel utility for combined telomere-specific FISH and flow cytometry (Flow FISH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1467.

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

Telomeres progressively shorten with each cell division, leading to critically short telomeres during tumorigenesis. While most cancers activate telomerase to maintain their telomeres, a subset of cancers utilize a recombination-based process known as alternative lengthening of telomeres, or ALT. ALT has been widely characterized mostly in the context of immortalized cells, and characteristic features include ultrabright telomeric foci, colocalization of telomere foci with PML (ALT-associated PML bodies, or APBs), extrachromosomal telomeric DNA (e.g. c-circles), and overall telomere length heterogeneity. While the overall rate of ALT in cancer is low (roughly 5%, overall), the prevalence of ALT is concentrated in certain cancer subtypes, including gliomas. Gliomas employing ALT have a common genomic signature: inactivating mutation of ATRX, a chromatin-modifying protein that functions at the telomeres. In order to study the role of ATRX loss in the biology of ALT in cancer, we have characterized a panel of six human cell lines derived from pediatric high grade glioma, two of which are ALT(+) and lack functional ATRX. Furthermore, in four pediatric and three adult high grade glioma cell lines that endogenously express functional ATRX, we knocked out ATRX using CRISPR/Cas9n and knocked down ATRX expression using a stable shRNA-mediated approach. Knockout or knockdown of ATRX was successful in these seven glioma cell lines. Overall, knockout and/or knockdown of ATRX induced the formation of ultrabright telomeric foci, as assessed by telomere FISH, in two out of seven glioma cell lines. However, the effect of ATRX loss on formation of c-circles and APBs was more variable: the presence of ultrabright telomeric foci was not predictive of the presence of c-circles or ALT-associated PML bodies. These results indicate that ATRX loss, alone, is not necessarily sufficient to activate the ALT pathway in gliomas. Further examination of ALT-competent and ALT-resistant cell lines from our cohort will yield clues as to additional ALT-associated gene expression changes. In addition, our results suggest that previously identified hallmarks of ALT (notably c-circles and APBs) may be more variable in cancer cells compared to immortalized normal cell lines. The ALT pathway is an intriguing target for synthetic lethality in the cancer subtypes that employ it; our hope is to better understand the ALT phenotype in cancer so that we may effectively translate ALT-specific therapeutics to the clinic. Citation Format: Jacqueline A. Brosnan-Cashman, Ming Yuan, Anthony J. Rizzo, Kaylar M. Myers, Walter T. Barry, Christine Davis, David M. Esopi, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Context-dependent effects of ATRX loss on telomere biology in glioma 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 3465. doi:10.1158/1538-7445.AM2017-3465

Overcoming the end-replication problem is a major hurdle for cancer cells due to progressive telomere shortening that results from excessive cell division during tumorigenesis. While most cancers activate telomerase to maintain their telomere length, about five percent of cancers use a telomerase-independent telomere maintenance mechanism, termed alternative lengthening of telomeres (ALT). Despite its low overall frequency in cancer, ALT is enriched in several cancer subtypes, including pediatric high grade glioma (HGG). Previous work in our laboratory suggests that nearly half of pediatric HGG utilize the ALT mechanism. To date, therapeutic options are largely ineffective for children with HGG, reflected in the dismal five-year survival rate, which is less than 33 percent. Our goal is to better harness ALT as a clinical marker in pediatric HGG, specifically by identifying drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pediatric HGG cell lines. Two of the six cell lines in this panel display hallmarks of the ALT pathway. To assess the potential of ALT as a therapeutic biomarker in pediatric HGG, we measured cell viability in the presence of inhibitors, stratifying our analysis by ALT status. ALT is predicted to occur via a homologous-recombination-based mechanism. As such, we focused our attention on inhibitors of the DNA damage response, as well as agents that induce DNA damage. Treatment with inhibitors of DNA-PK, RAD51, and MRE11 did not result in significant differences in cell viability when stratified by ALT status, nor did treatment with temozolomide or hydroxyurea. However, inhibitors of ATR (VE-821, VE-822/VX-970, and AZD-6738) and CHK1 (MK-8776) led to significantly greater reductions in cell viability in ALT-positive cells. In order to further our understanding of the mechanism through which ATR inhibition preferentially targets ALT-positive cell lines, we examined the effect of these inhibitors on ALT-specific properties, such as the presence of ultrabright telomeric foci and extrachromosomal telomeric DNA (c-circles). Preliminary results indicate that ATR inhibition does not diminish these ALT-associated properties and, therefore, the differential sensitivity to these agents is unlikely to be due to directly blocking the ALT telomere maintenance mechanism. Continued study of the effect of ATR/CHK1 inhibition on ALT-positive cells will yield further insight into the mechanism of ALT-specific toxicity. Overall, we have identified ATR/CHK1 as a promising pathway to target for ALT-positive pediatric HGG. Our goal is to better understand the relationship between ATR/CHK1 signaling and ALT in order to effectively translate this observation to the clinic, both for pediatric HGG and for other ALT-positive cancers. Citation Format: Jacqueline A. Brosnan-Cashman, Anthony J. Rizzo, Ezgi Goger, Kaylar M. Myers, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition [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 3466. doi:10.1158/1538-7445.AM2017-3466

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.