DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate.

BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

MYC drives overexpression of telomerase RNA (hTR/TERC) in prostate cancer.

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate-limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high-grade prostatic intraepithelial neoplasia or PIN) and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma and that its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeting the human androgen receptor gene with platinated triplex-forming oligonucleotides.

Platinum-derivatized homopyrimidine triplex-forming oligonucleotides (Pt-TFOs) consisting of 2'-O-methyl-5-methyluridine, 2'-O-methyl-5-methylcytidine, and a single 3'-N7-trans-chlorodiammine platinum(II)-2'-deoxyguanosine were designed to cross-link to the transcribed strand at four different sequences in the human androgen receptor (AR) gene. Fluorescence microscopy showed that a fluorescein-tagged Pt-TFO localizes in both the cytoplasm and nucleus when it is transfected into LAPC-4 cells, a human prostate cancer cell line, using Lipofectamine 2000. A capture assay employing streptavidin-coated magnetic beads followed by polymerase chain reaction (PCR) amplification was used to demonstrate that 5'-biotin-conjugated Pt-TFOs cross-link in vitro to their four designated AR gene targets in genomic DNA extracted from LAPC-4 cells. Similarly, the capture assay was used to examine cross-linking between the 5'-biotin-conjugated Pt-TFOs and the AR gene in LAPC-4 cells in culture. Three of the four Pt-TFOs cross-linked to their designated target, suggesting that different regions of the AR gene are not uniformly accessible to Pt-TFO cross-linking. LAPC-4 cells were transfected with fluorescein-tagged Pt-TFO or a control oligonucleotide that does not bind or cross-link to AR DNA. The levels of AR mRNA in highly fluorescent cells isolated by fluorescence-activated cell sorting were determined by RT-qPCR, and the levels of AR protein were monitored by immunofluorescence microscopy. Decreases in mRNA and protein levels of 40 and 30%, respectively, were observed for fluorescein-tagged Pt-TFO versus control treated cells. Although the levels of knockdown of AR mRNA and protein were modest, the results suggest that Pt-TFOs hold potential as agents for controlling gene expression by cross-linking to DNA and disrupting transcription.

The TERT Promoter is Polycomb-Repressed in Neuroblastoma Cells with Long Telomeres.

Acquiring a telomere maintenance mechanism is a hallmark of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has long telomeres and utilizes the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres. Here, we show, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5 kb. The TERT locus shows active chromatin marks with low enrichment for the repressive mark, H3K27me3. MYCN, a commonly amplified oncogene in neuroblstoma, binds to the promoter and induces TERT expression. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect (TPE). Inhibiting H3K27 methylation by three different EZH2 inhibitors induced the expression of TERT in cell lines with long telomeres and H3K27me3 marks in the promoter region. EZH2 inhibition facilitated MYCN binding to the TERT promoter in neuroblastoma cells with long telomeres. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres. SIGNIFICANCE: The epigenetic landscape of the TERT locus is unique in neuroblastoma. The DNA at the TERT locus, unlike other cancer cells and similar to normal cells, are hypomethylated in telomerase-positive neuroblastoma cells. The TERT locus is repressed by polycomb repressive complex-2 complex in neuroblastoma cells that have long telomeres and do not express TERT. Long telomeres in neuroblastoma cells are also associated with repressive chromatin states at the chromosomal termini, suggesting TPE.

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer.

How prostate cancer cells and their precursors mediate changes in the tumor microenvironment (TME) to drive prostate cancer progression is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we perform extensive single-cell RNA-sequencing (scRNA-seq) and molecular pathology of the comparative biology between human prostate cancer and key stages in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues reveal that cancer cell-intrinsic activation of MYC signaling is a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Cell communication network and pathway analyses in GEMMs show that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogram the TME during carcinogenesis, leading to a convergence of cell state alterations in neighboring epithelial, immune, and fibroblast cell types that parallel key findings in human prostate cancer.

Prognostic and therapeutic potential of senescent stromal fibroblasts in prostate cancer.

The stromal component of the tumour microenvironment in primary and metastatic prostate cancer can influence and promote disease progression. Within the prostatic stroma, fibroblasts are one of the most prevalent cell types associated with precancerous and cancerous lesions; they have a vital role in the structural composition, organization and integrity of the extracellular matrix. Fibroblasts within the tumour microenvironment can undergo cellular senescence, which is a stable arrest of cell growth and a phenomenon that is emerging as a recognized hallmark of cancer. Supporting the idea that cellular senescence has a pro-tumorigenic role, a subset of senescent cells exhibits a senescence-associated secretory phenotype (SASP), which, along with increased inflammation, can promote prostate cancer cell growth and survival. These cellular characteristics make targeting senescent cells and/or modulating SASP attractive as a potential preventive or therapeutic option for prostate cancer.

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer.

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

Inhibition of transcription by platinated triplex-forming oligonucleotides.

Platinated triplex-forming oligonucleotides (TFOs) consisting of 2'-methoxythymidine and 2'-methoxy-5-methylcytidine and an N-7 platinated deoxyguanosine ((Pt)G) at the 5'-((Pt)G-TFO), 3'-(TFO-G(Pt)), or 3'- and 5'-((Pt)G-TFO-G(Pt)) ends of the TFO form mono-((Pt)G-TFO and TFO-G(Pt)) and interstrand ((Pt)G-TFO-G(Pt)) cross-links with target DNA as a result of reaction of the (Pt)G with guanines adjacent to the homopurine TFO binding site in the target. The extent of cross-linking is greatest when the (Pt)G is located on the 3' end of the TFO and the target guanine is on the same strand as the TFO binding site. Multiple, contiguous deoxyguanosines in the TFO binding site or a cytosine adjacent to the G(Pt) of the TFO significantly reduce cross-linking. DNA reporter plasmids in which platinated TFOs were cross-linked at a site in the transcribed region between a CMV promoter and a luciferase reporter gene were transfected into Chinese hamster ovary cells, and luciferase expression was compared with that for the corresponding non-cross-linked plasmid. Luciferase expression was inhibited 95 % when TFO-G(Pt) was bound and cross-linked to the transcribed strand, demonstrating that the cross-linked TFO was able to block transcription elongation. Further inhibition (99 %) was observed in nucleotide excision repair (NER) deficient cells, suggesting that NER may repair this lesion. The 3'-G(Pt) group of TFO-G(Pt) protects the TFO from degradation by exonucleases found in mammalian serum. Taken together, these results suggest that platinated TFOs of the type TFO-G(Pt) may find applications as agents for suppressing DNA transcription and consequently inhibiting gene expression in mammalian cells.

Telomere lengths differ significantly between small-cell neuroendocrine prostate carcinoma and adenocarcinoma of the prostate.

Small-cell neuroendocrine carcinoma (SCNC) of the prostate is an aggressive subtype with frequent TP53 mutation and RB1 inactivation; however, the molecular phenotype remains an area of investigation. Here, we compared telomere lengths in prostatic SCNC and usual-type prostatic adenocarcinoma (AdCa). We studied 32 cases of prostatic SCNC (including 11 cases with concurrent AdCa) and 347 cases of usual-type AdCa on tissue microarrays. Telomere lengths in tumor cells were qualitatively compared with those in normal cells using a telomere-specific fluorescence in situ hybridization assay. ERG, PTEN, and TP53 status were assessed in a proportion of cases using genetically validated immunohistochemistry protocols. Clinicopathological and molecular characteristics of cases were compared between the telomere groups using the chi-square test.A significantly higher proportion of prostatic SCNC cases (50%, 16/32) showed normal/long telomeres compared with AdCa cases (11%, 39/347; P < 0.0001). In 82% (9/11) of cases with concurrent SCNC and AdCa, the paired components were concordant for telomere length status. Among AdCa cases, the proportion of cases with normal/long telomeres significantly increased with increasing tumor grade group (P = 0.01) and pathologic stage (P = 0.02). Cases with normal/long telomeres were more likely to be ERG positive (P = 0.04) and to have TP53 missense mutation (P = 0.01) than cases with short telomeres.Normal or long telomere lengths are significantly more common in prostatic SCNC than in AdCa and are similar between concurrent SCNC and AdCa tumors, supporting a common origin. Among AdCa cases, longer telomere lengths are significantly associated with high-risk pathologic and molecular features.

Telomere alterations in neurofibromatosis type 1-associated solid tumors.

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 < 0.0001). In the NF1-MPNST group, overall survival was superior for patients with tumors with short telomeres (p = 0.003). ALT occurs in a subset of NF1-associated solid tumors and is usually restricted to malignant subsets. In contrast, alterations in telomere lengths are more prevalent than ALT.

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

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

Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors.

Most pancreatic neuroendocrine tumors (PNETs) do not produce excess hormones and are therefore considered 'non-functional'1-3. As clinical behaviors vary widely and distant metastases are eventually lethal2,4, biological classifications might guide treatment. Using enhancer maps to infer gene regulatory programs, we find that non-functional PNETs fall into two major subtypes, with epigenomes and transcriptomes that partially resemble islet α- and ß-cells. Transcription factors ARX and PDX1 specify these normal cells, respectively5,6, and 84% of 142 non-functional PNETs expressed one or the other factor, occasionally both. Among 103 cases, distant relapses occurred almost exclusively in patients with ARX+PDX1- tumors and, within this subtype, in cases with alternative lengthening of telomeres. These markedly different outcomes belied similar clinical presentations and histology and, in one cohort, occurred irrespective of MEN1 mutation. This robust molecular stratification provides insight into cell lineage correlates of non-functional PNETs, accurately predicts disease course and can inform postoperative clinical decisions.

Publisher Correction: Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner.

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.

Alternative lengthening of telomeres and ATRX/DAXX loss can be reliably detected in FNAs of pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PanNETs) frequently use the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. ALT is strongly correlated with α thalassemia-mental retardation, X linked (ATRX), and death domain-associated protein 6 (DAXX) alterations and a poor prognosis in patients with primary PanNET. Because fine-needle aspiration (FNA) is a noninvasive way to sample tumors, the authors evaluated whether they could accurately detect ALT and loss of ATRX/DAXX in a primary PanNET cohort of FNAs. METHODS: All preoperative FNA cytology cases (2005-2016) with adequate remnant FNA cell block material were assessed for ALT by telomere-specific fluorescence in situ hybridization and for ATRX and DAXX protein expression by immunohistochemistry. For 21 patients who underwent tumor resection, the resected specimen also was assessed to determine the concordance between the FNA and surgical specimens. RESULTS: In the primary PanNET cohort of 65 FNAs, ALT was detected in 15 specimens (23%). Although all ATRX-negative and DAXX-negative tumors were ALT-positive, 3 of 14 (21%) ALT-positive tumors did not exhibit nuclear loss of either ATRX or DAXX. The ALT-positive tumors were associated with larger radiographic size (4.9 vs 2.4 cm, on average; P < .05) and higher grade (P < .05). Overall, there was 100% concordance in ALT status and ATRX/DAXX immunohistochemistry results between the FNA and surgical specimens. CONCLUSIONS: Both ALT and loss of ATRX/DAXX can be accurately performed on FNA specimens with adequate material. Because ALT is a fundamental mechanism of pathogenesis, the ability to determine ALT in small biospecimens has implications for the design of clinical trials. Cancer Cytopathol 2017;125:544-51. © 2017 American Cancer Society.