CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma.

p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.

BEND3 safeguards pluripotency by repressing differentiation-associated genes.

BEN domain-containing proteins are emerging rapidly as an important class of factors involved in modulating gene expression, yet the molecular basis of how they regulate chromatin function and transcription remains to be established. BEND3 is a quadruple BEN domain-containing protein that associates with heterochromatin and functions as a transcriptional repressor. We find that BEND3 is highly expressed in pluripotent cells, and the induction of differentiation results in the down-regulation of BEND3. The removal of BEND3 from pluripotent cells results in cells exhibiting upregulation of the differentiation-inducing gene expression signature. We find that BEND3 binds to the promoters of differentiation-associated factors and key cell cycle regulators, including CDKN1A, encoding the cell cycle inhibitor p21, and represses the expression of differentiation-associated genes by enhancing H3K27me3 decoration at these promoters. Our results support a model in which transcription repression mediated by BEND3 is essential for normal development and to prevent differentiation.

Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.

During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association with two phospho-isoforms of the helicase component MCM2. During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin. In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation. In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

Primary Founder Mutations in the PRKDC Gene Increase Tumor Mutation Load in Colorectal Cancer.

The clonal composition of a malignant tumor strongly depends on cellular dynamics influenced by the asynchronized loss of DNA repair mechanisms. Here, our aim was to identify founder mutations leading to subsequent boosts in mutation load. The overall mutation burden in 591 colorectal cancer tumors was analyzed, including the mutation status of DNA-repair genes. The number of mutations was first determined across all patients and the proportion of genes having mutation in each percentile was ranked. Early mutations in DNA repair genes preceding a mutational expansion were designated as founder mutations. Survival analysis for gene expression was performed using microarray data with available relapse-free survival. Of the 180 genes involved in DNA repair, the top five founder mutations were in PRKDC (n = 31), ATM (n = 26), POLE (n = 18), SRCAP (n = 18), and BRCA2 (n = 15). PRKDC expression was 6.4-fold higher in tumors compared to normal samples, and higher expression led to longer relapse-free survival in 1211 patients (HR = 0.72, p = 4.4 × 10-3). In an experimental setting, the mutational load resulting from UV radiation combined with inhibition of PRKDC was analyzed. Upon treatments, the mutational load exposed a significant two-fold increase. Our results suggest PRKDC as a new key gene driving tumor heterogeneity.

A snapshot of 3649 Web-based services published between 1994 and 2017 shows a decrease in availability after 2 years.

BACKGROUND: The long-term availability of online Web services is of utmost importance to ensure reproducibility of analytical results. However, because of lack of maintenance following acceptance, many servers become unavailable after a short period of time. Our aim was to monitor the accessibility and the decay rate of published Web services as well as to determine the factors underlying trends changes. METHODS: We searched PubMed to identify publications containing Web server-related terms published between 1994 and 2017. Automatic and manual screening was used to check the status of each Web service. Kruskall-Wallis, Mann-Whitney and Chi-square tests were used to evaluate various parameters, including availability, accessibility, platform, origin of authors, citation, journal impact factor and publication year. RESULTS: We identified 3649 publications in 375 journals of which 2522 (69%) were currently active. Over 95% of sites were running in the first 2 years, but this rate dropped to 84% in the third year and gradually sank afterwards (P < 1e-16). The mean half-life of Web services is 10.39 years. Working Web services were published in journals with higher impact factors (P = 4.8e-04). Services published before the year 2000 received minimal attention. The citation of offline services was less than for those online (P = 0.022). The majority of Web services provide analytical tools, and the proportion of databases is slowly decreasing. Conclusions. Almost one-third of Web services published to date went out of service. We recommend continued support of Web-based services to increase the reproducibility of published results.

TPMCalculator: one-step software to quantify mRNA abundance of genomic features.

SUMMARY: The quantification of RNA sequencing (RNA-seq) abundance using a normalization method that calculates transcripts per million (TPM) is a key step to compare multiple samples from different experiments. TPMCalculator is a one-step software to process RNA-seq alignments in BAM format and reports TPM values, raw read counts and feature lengths for genes, transcripts, exons and introns. The program describes the genomic features through a model generated from the gene transfer format file used during alignments reporting of the TPM values and the raw read counts for each feature. In this paper, we show the correlation for 1256 samples from the TCGA-BRCA project between TPM and FPKM reported by TPMCalculator and RSeQC. We also show the correlation for raw read counts reported by TPMCalculator, HTSeq and featureCounts. AVAILABILITY AND IMPLEMENTATION: TPMCalculator is freely available at https://github.com/ncbi/TPMCalculator. It is implemented in C++14 and supported on Mac OS X, Linux and MS Windows. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An integrative bioinformatics approach reveals coding and non-coding gene variants associated with gene expression profiles and outcome in breast cancer molecular subtypes.

BACKGROUND: Sequence variations in coding and non-coding regions of the genome can affect gene expression and signalling pathways, which in turn may influence disease outcome. METHODS: In this study, we integrated somatic mutations, gene expression and clinical data from 930 breast cancer patients included in the TCGA database. Genes associated with single mutations in molecular breast cancer subtypes were identified by the Mann-Whitney U-test and their prognostic value was evaluated by Kaplan-Meier and Cox regression analyses. Results were confirmed using gene expression profiles from the Metabric data set (n = 1988) and whole-genome sequencing data from the TCGA cohort (n = 117). RESULTS: The overall mutation rate in coding and non-coding regions were significantly higher in ER-negative/HER2-negative tumours (P = 2.8E-03 and P = 2.4E-07, respectively). Recurrent sequence variations were identified in non-coding regulatory regions of several cancer-associated genes, including NBPF1, PIK3CA and TP53. After multivariate regression analysis, gene signatures associated with three coding mutations (CDH1, MAP3K1 and TP53) and two non-coding variants (CRTC3 and STAG2) in cancer-related genes predicted prognosis in ER-positive/HER2-negative tumours. CONCLUSIONS: These findings demonstrate that sequence alterations influence gene expression and oncogenic pathways, possibly affecting the outcome of breast cancer patients. Our data provide potential opportunities to identify non-coding variations with functional and clinical relevance in breast cancer.

KRAS driven expression signature has prognostic power superior to mutation status in non-small cell lung cancer.

KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC). However, the prognostic role of KRAS mutation status in NSCLC still remains controversial. We hypothesize that the expression changes of genes affected by KRAS mutation status will have the most prominent effect and could be used as a prognostic signature in lung cancer. We divided NSCLC patients with mutation and RNA-seq data into KRAS mutated and wild type groups. Mann-Whitney test was used to identify genes showing altered expression between these cohorts. Mean expression of the top five genes was designated as a "transcriptomic fingerprint" of the mutation. We evaluated the effect of this signature on clinical outcome in 2,437 NSCLC patients using univariate and multivariate Cox regression analysis. Mutation of KRAS was most common in adenocarcinoma. Mutation status and KRAS expression were not correlated to prognosis. The transcriptomic fingerprint of KRAS include FOXRED2, KRAS, TOP1, PEX3 and ABL2. The KRAS signature had a high prognostic power. Similar results were achieved when using the second and third set of strongest genes. Moreover, all cutoff values delivered significant prognostic power (p < 0.01). The KRAS signature also remained significant (p < 0.01) in a multivariate analysis including age, gender, smoking history and tumor stage. We generated a "surrogate signature" of KRAS mutation status in NSCLC patients by computationally linking genotype and gene expression. We show that secondary effects of a mutation can have a higher prognostic relevance than the primary genetic alteration itself.

Predictors of Chemosensitivity in Triple Negative Breast Cancer: An Integrated Genomic Analysis.

BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients. METHODS AND FINDINGS: We performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort. CONCLUSIONS: The comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.

Applied models and molecular characteristics of small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive type of cancer frequently diagnosed with metastatic spread, rendering it surgically unresectable for the majority of patients. Although initial responses to platinum-based therapies are often observed, SCLC invariably relapses within months, frequently developing drug-resistance ultimately contributing to short overall survival rates. Recently, SCLC research aimed to elucidate the dynamic changes in the genetic and epigenetic landscape. These have revealed distinct subtypes of SCLC, each characterized by unique molecular signatures. The recent understanding of the molecular heterogeneity of SCLC has opened up potential avenues for precision medicine, enabling the development of targeted therapeutic strategies. In this review, we delve into the applied models and computational approaches that have been instrumental in the identification of promising drug candidates. We also explore the emerging molecular diagnostic tools that hold the potential to transform clinical practice and patient care.

Genome-wide Mapping of Topoisomerase Binding Sites Suggests Topoisomerase 3α (TOP3A) as a Reader of Transcription-Replication Conflicts (TRC).

Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and topological problems. Here we asked which topoisomerase(s) is/are the best candidate(s) for sensing TRC. Genome-wide topoisomerase binding sites were mapped in parallel for all the nuclear topoisomerases (TOP1, TOP2A, TOP2B, TOP3A and TOP3B). To increase the signal to noise ratio (SNR), we used ectopic expression of those topoisomerases in H293 cells followed by a modified CUT&Tag method. Although each topoisomerase showed distinct binding patterns, all topoisomerase binding signals positively correlated with gene transcription. TOP3A binding signals were suppressed by DNA replication inhibition. This was also observed but to a lesser extent for TOP2A and TOP2B. Hence, we propose the involvement of TOP3A in sensing both head-on TRCs (HO-TRCs) and co-directional TRCs (CD-TRCs). In which case, the TOP3A signals appear concentrated within the promoters and first 20 kb regions of the 5' -end of genes, suggesting the prevalence of TRCs and the recruitment of TOP3A in the 5'-regions of transcribed and replicated genes.

Sarcoma_CellminerCDB: A tool to interrogate the genomic and functional characteristics of a comprehensive collection of sarcoma cell lines.

Sarcomas are a diverse group of rare malignancies composed of multiple different clinical and molecular subtypes. Due to their rarity and heterogeneity, basic, translational, and clinical research in sarcoma has trailed behind that of other cancers. Outcomes for patients remain generally poor due to an incomplete understanding of disease biology and a lack of novel therapies. To address some of the limitations impeding preclinical sarcoma research, we have developed Sarcoma_CellMinerCDB, a publicly available interactive tool that merges publicly available sarcoma cell line data and newly generated omics data to create a comprehensive database of genomic, transcriptomic, methylomic, proteomic, metabolic, and pharmacologic data on 133 annotated sarcoma cell lines. The reproducibility, functionality, biological relevance, and therapeutic applications of Sarcoma_CellMinerCDB described herein are powerful tools to address and generate biological questions and test hypotheses for translational research. Sarcoma_CellMinerCDB (https://discover.nci.nih.gov/SarcomaCellMinerCDB) aims to contribute to advancing the preclinical study of sarcoma.

Genomic alterations and transcriptional phenotypes in circulating tumor DNA and matched metastatic tumor.

Background: Profiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), a cancer whose aggressive clinical course making it exceedingly challenging to obtain tumor biopsies. Methods: Here, a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC, we study cfDNA low pass whole genome (0.1X coverage) and exome (130X) sequencing in comparison with time-point matched tumor, characterized using exome and transcriptome sequencing. Results: Direct comparison of cfDNA versus tumor biopsy reveals that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not found in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Genomic sequencing coverage of plasma DNA fragments around transcription start sites shows distinct treatment-related changes and captures the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors, allowing prediction of SCLC neuroendocrine phenotypes and treatment responses. Conclusions: These findings have important implications for non-invasive stratification and subtype-specific therapies for patients with SCLC, now treated as a single disease.

Staufen1 Represses the FOXA1-Regulated Transcriptome by Destabilizing FOXA1 mRNA in Colorectal Cancer Cells.

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.

D-type cyclins regulate DNA mismatch repair in the G1 and S phases of the cell cycle, maintaining genome stability.

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S.

Metastatic organotropism in small cell lung cancer.

Metastasis is the leading cause of cancer-related deaths, yet its regulatory mechanisms are not fully understood. Small-cell lung cancer (SCLC) is the most metastatic form of lung cancer, with most patients presenting with widespread disease, making it an ideal model for studying metastasis. However, the lack of suitable preclinical models has limited such studies. We utilized well-annotated rapid autopsy-derived tumors to develop xenograft models that mimic key features of SCLC, including histopathology, rapid and widespread development of metastasis to the liver, brain, adrenal, bone marrow, and kidneys within weeks, and response to chemotherapy. By integrating in vivo lineage selection with comprehensive transcriptomic and epigenomic analyses, we identified critical cellular programs driving metastatic organotropism to the liver and brain, the most common sites of SCLC metastasis. Our findings reveal the key role of nuclear-cytoskeletal interactions in SCLC liver metastasis. Specifically, the loss of the nuclear envelope protein lamin A/C, encoded by the LMNA gene, increased nuclear deformability and significantly increased the incidence of liver metastasis. Human liver metastases exhibited reduced LMNA expression compared to other metastatic sites, correlating with poorer patient outcomes and increased mortality. This study introduces novel preclinical models for SCLC metastasis and highlights pathways critical for organ-specific metastasis, offering new avenues for the development of targeted therapies to prevent or treat metastatic disease.

Microenvironment shapes small-cell lung cancer neuroendocrine states and presents therapeutic opportunities.

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intratumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the cell-extrinsic drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the TME exhibit substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAFs) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLC's adaptable nature, opening possibilities for reprogramming the TME-tumor communications that shape SCLC tumor states.

Matrin3 regulates mitotic spindle dynamics by controlling alternative splicing of CDC14B.

Matrin3 is an RNA-binding protein that regulates diverse RNA-related processes, including mRNA splicing. Although Matrin3 has been intensively studied in neurodegenerative diseases, its function in cancer remains unclear. Here, we report Matrin3-mediated regulation of mitotic spindle dynamics in colorectal cancer (CRC) cells. We comprehensively identified RNAs bound and regulated by Matrin3 in CRC cells and focused on CDC14B, one of the top Matrin3 targets. Matrin3 knockdown results in increased inclusion of an exon containing a premature termination codon in the CDC14B transcript and simultaneous down-regulation of the standard CDC14B transcript. Knockdown of CDC14B phenocopies the defects in mitotic spindle dynamics upon Matrin3 knockdown, and the elongated and misoriented mitotic spindle observed upon Matrin3 knockdown are rescued upon overexpression of CDC14B, suggesting that CDC14B is a key downstream effector of Matrin3. Collectively, these data reveal a role for the Matrin3/CDC14B axis in control of mitotic spindle dynamics.

SIRT1 Prevents R-Loops during Chronological Aging by Modulating DNA Replication at rDNA Loci.

In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication, and repair. Our previous studies have demonstrated that SIRT1 binds replication origins and inhibits replication initiation from a group of potential initiation sites (dormant origins). We studied the effects of aging and SIRT1 activity on replication origin usage and the incidence of transcription-replication collisions (creating R-loop structures) in adult human cells obtained at different time points during chronological aging and in cancer cells. In primary, untransformed cells, SIRT1 activity declined and the prevalence of R-loops rose with chronological aging. Both the reduction in SIRT1 activity and the increased abundance of R-loops were also observed during the passage of primary cells in culture. All cells, regardless of donor age or transformation status, reacted to the short-term, acute chemical inhibition of SIRT1 with the activation of excessive replication initiation events coincident with an increased prevalence of R-loops. However, cancer cells activated dormant replication origins, genome-wide, during long-term proliferation with mutated or depleted SIRT1, whereas, in primary cells, the aging-associated SIRT1-mediated activation of dormant origins was restricted to rDNA loci. These observations suggest that chronological aging and the associated decline in SIRT1 activity relax the regulatory networks that protect cells against excess replication and that the mechanisms protecting from replication-transcription collisions at the rDNA loci manifest as differentially enhanced sensitivities to SIRT1 decline and chronological aging.