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

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Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells Mindy K Published OnlineFirst October 14, 2019; DOI: 10.1158/1541-7786.MCR-19-0654 Genome Maintenance Molecular Cancer Research Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells Mindy K. Graham1, Jiyoung Kim1, Joseph Da1, Jacqueline A. Brosnan-Cashman1, Anthony Rizzo1, Javier A. Baena Del Valle1, Lionel Chia1, Michael Rubenstein2, Christine Davis1, Qizhi Zheng1, Leslie Cope3, Michael Considine3, Michael C. Haffner1, Angelo M. De Marzo1,3,4, Alan K. Meeker1,3,4, and Christopher M. Heaphy1,3 Abstract A key hallmark of cancer, unlimited replication, requires LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient cancer cells to evade both replicative senescence and potentially to induce multiple ALT-associated hallmarks, including the lethal chromosomal instability induced by telomere dysfunc- presence of ALT-associated promyelocytic leukemia bodies tion. The majority of cancers overcome these critical barriers by (APB), extrachromosomal telomere C-circles, and dramatic upregulating telomerase, a telomere-specific reverse transcrip- telomere length heterogeneity. However, telomerase activity tase. However, a subset of cancers maintains telomere lengths was still present in these ATRXKO cells. Telomerase activity was by the telomerase-independent Alternative Lengthening of subsequently crippled in these LAPC-4 ATRXKO cells by intro- Telomeres (ALT) pathway. The presence of ALT is strongly ducing mutations in the TERC locus, the essential RNA com- associated with recurrent cancer-specific somatic inactivating ponent of telomerase. These LAPC-4 ATRXKO TERCmut cells mutations in the ATRX-DAXX chromatin-remodeling complex. continued to proliferate long-term and retained ALT-associated Here, we generate an ALT-positive adenocarcinoma cell line hallmarks, thereby demonstrating their reliance on the ALT following functional inactivation of ATRX and telomerase in a mechanism for telomere maintenance. telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase Implications: These prostate cancer cell line models provide a into two prostate cancer cell lines, LAPC-4 (derived from a unique system to explore the distinct molecular alterations that lymph node metastasis) and CWR22Rv1 (sourced from a occur upon induction of ALT, and may be useful tools to screen xenograft established from a primary prostate cancer). In for ALT-specific therapies. Introduction incomplete DNA replication in the lagging strand (2). Once telo- meres reach a critical length, normal cells undergo p53-dependent Telomeres are nucleoprotein complexes that consist of a repet- replicative senescence or apoptosis (3). While there are rare excep- itive hexameric DNA sequence, 50-TTAGGG/30-AATCCC, that are tions without a known telomere maintenance mechanism (4, 5), bound by the shelterin protein complex. Located at the ends of telomere maintenance is required for most cancers to maintain eukaryotic chromosomes, telomeres maintain genomic stability unlimited replication while evading replicative senescence or apo- and integrity by preventing exonucleolytic degradation and recog- ptosis (6). The vast majority of cancers upregulate telomerase, a nition of the chromosomal ends as double stranded breaks by DNA telomere-specific reverse transcriptase (7); however, 10%–15% of damage response (DDR) proteins (1). Following each round of cell cancers lack telomerase activity (7, 8). division, telomeres progressively shorten as a direct result of A subset of telomerase-negative cancers maintains their telo- mere lengths by a telomere-specific mechanism of homology- directed repair called Alternative Lengthening of Telomeres (ALT; 1 Department of Pathology, Johns Hopkins University School of Medicine, Balti- refs. 9–11). The mechanism underlying ALT generates unique 2 more, Maryland. Department of Biological Sciences, University of Maryland molecular characteristics that readily distinguish ALT-positive and Baltimore County, Baltimore, Maryland. 3Department of Oncology Johns Hop- kins University School of Medicine, Baltimore, Maryland. 4Department of Urol- ALT-negative cancers. For example, ALT-positive cancers display ogy, Johns Hopkins University School of Medicine, Baltimore, Maryland. dramatic telomere length heterogeneity, manifesting both as cell- to-cell variation and intracellular heterogeneity (9). ALT-positive Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). cancers also have ALT-associated promyelocytic leukemia (PML) bodies, called APBs. These unique nuclear structures contain Corresponding Author: Christopher M. Heaphy Johns Hopkins University School donut-shaped PML protein bodies that colocalize with telomeric of Medicine, 411 N. Caroline St., Bond St. Bldg B301, Baltimore, MD 21231. Phone: 443-287-4730; Fax. 410-592-5158; E-mail: [email protected] DNA, the shelterin components TRF1 and TRF2, and proteins involved in DNA synthesis and recombination (12, 13). In Mol Cancer Res 2019;17:1–12 addition, ALT-positive cancers are enriched for C-circles, extra- doi: 10.1158/1541-7786.MCR-19-0654 chromosomal circular DNA comprised of single-stranded C-rich Ó2019 American Association for Cancer Research. telomere DNA sequence partially overlapped by linear www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 14, 2019; DOI: 10.1158/1541-7786.MCR-19-0654 Graham et al. complementary G-rich telomere DNA (14). Sequencing studies of (Sigma), 1% of mixture of penicillin (10,000 U/mL) and strep- molecularly characterized ALT-positive cancers from our group, tomycin (10,000 mg/mL; Quality Biological), and 1 nmol/L of and others, identified a robust association between ALT and R1881. CWR22Rv1 and PC3 were cultured in RPMI1640 (Gibco) mutations in a-thalassemia/mental retardation X-linked protein supplemented with 10% FBS and 1% penicillin/streptomycin. (ATRX) and death domain–associated protein (DAXX) in multi- U2OS was cultured in DMEM, high glucose (DMEM, Gibco) ple tumor types, including pancreatic neuroendocrine tumors supplemented with 10% FBS and 1% penicillin/streptomycin. (PanNET), sarcomas, and tumors of the central nervous All cell lines were submitted to the Genetic Resources Core Facility system (15–17). As a complex, ATRX/DAXX recognizes H3K9me3 at Johns Hopkins for Mycoplasma detection and cell line authen- in chromatin and deposits histone variant H3.3 at repetitive tication by short tandem repeat (STR) profiling using the Gene- sequences. This chromatin-remodeling function of ATRX/DAXX Print 10 kit (Promega, June 2018). is important for heterochromatin maintenance at repetitive sequences, including telomeres (18, 19). More recently, inactivat- ATRX CRISPR genome editing ing mutations in the ATP-dependent annealing helicase, SMAR- As described previously, two CRISPR Cas9 nickase guide RNAs CAL1, were also found to be tightly associated with ALT in a subset (Supplementary Table S1) were designed to target exon 9 of ATRX of ATRX and DAXX wild-type glioblastomas (20). using CRISPR Design (crispr.mit.edu, Supplementary Fig. S2). The majority of established ALT-positive cancer cell lines have The gRNAs were cloned into the GFP-expressing Cas9n plasmid, inactivated ATRX, although there are some recent excep- PX461, a gift from Feng Zhang (Addgene #48140; ref. 33). Lipo- tions (20–23). Previous attempts to induce ALT through knock- fectamine 3000 (Thermo Fisher Scientific) was used to transfect down or knockout of ATRX have largely been unsuccess- either empty vector PX461 or cotransfect both ATRX gRNA1- ful (21, 23–25). However, in a context-dependent manner, genet- PX461 and ATRX gRNA 2-PX461 into LAPC-4 and CWR22Rv1 ic knockout of ATRX or SMARCAL1 in some telomerase-positive cells. GFP-positive cells were sorted by FACS after 48 hours and glioma cell lines has induced multiple hallmarks of ALT (20, 26). 1,000 cells were plated in 150-mm dishes. Cell colonies were Thus, a constellation of genetic and epigenetic changes may be isolated using cloning cylinders (Sigma) and screened prelim- gatekeepers for permitting ALT. Interestingly, the combination of inarily for ATRX protein by immunostaining. Promising clones knocking down ATRX, knocking out DAXX, and introducing a were subsequently validated by Western blotting and Sanger mutation in TPP1 (that suppresses telomerase activity and sequencing. induces telomere-specific DNA damage) was sufficient to activate ALT in the telomerase-positive fibrosarcoma cell line HTC75 (27). CRISPR genome editing of the TERC locus Strategies to cripple telomerase via knocking out TERC in telo- A CRISPR plasmid cloned with the TERC gRNA-1 (Supplemen- merase-positive cell lines have been successful in activating ALT at tary Table S1) cloned into plasmid PX458 was a kind gift extremely low frequency in the spontaneously immortalized from Jaewon Min and colleagues (Department of Cell Biology, human lung fibroblast cell line SW39 and the lung carcinoma University of Texas Southwestern Medical Center; ref. 34). The cell line H1299 (28). original vector was a gift from Feng Zhang (Addgene #48138; We previously identified a prostate cancer case where a chro- ref. 35). Lipofectamine 3000 (Thermo Fisher Scientific) was used mosomal inversion disrupting ATRX was found in multiple to transfect TERC1-gRNA-PX458 in LAPC-4 ATRXKO cells. GFP- distant metastases, but not in
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