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.

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.

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.

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.

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.

Extrachromosomal DNA Amplification Contributes to Small Cell Lung Cancer Heterogeneity and Is Associated with Worse Outcomes.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. Oncogenic MYC amplifications drive SCLC heterogeneity, but the genetic mechanisms of MYC amplification and phenotypic plasticity, characterized by neuroendocrine and nonneuroendocrine cell states, are not known. Here, we integrate whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as a primary source of SCLC oncogene amplifications and driver fusions. ecDNAs bring to proximity enhancer elements and oncogenes, creating SCLC transcription-amplifying units, driving exceptionally high MYC gene dosage. We demonstrate that cell-free nucleosome profiling can noninvasively detect ecDNA amplifications in plasma, facilitating its genome-wide interrogation in SCLC and other cancers. Altogether, our work provides the first comprehensive map of SCLC ecDNA and describes a new mechanism that governs MYC-driven SCLC heterogeneity. ecDNA-enabled transcriptional flexibility may explain the significantly worse survival outcomes of SCLC harboring complex ecDNA amplifications. SIGNIFICANCE: MYC drives SCLC progression, but the genetic basis of MYC-driven SCLC evolution is unknown. Using SCLC as a paradigm, we report how ecDNA amplifications function as MYC-amplifying units, fostering tumor plasticity and a high degree of tumor heterogeneity. This article is highlighted in the In This Issue feature, p. 799.

Context-Dependent Function of Long Noncoding RNA PURPL in Transcriptome Regulation during p53 Activation.

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

Integrative epigenomic analyses of small cell lung cancer cells demonstrates the clinical translational relevance of gene body methylation.

DNA methylation is a key regulator of gene expression and a clinical therapeutic predictor. We examined global DNA methylation beyond the generally used promoter areas in human small cell lung cancer (SCLC) and find that gene body methylation is a robust positive predictor of gene expression. Combining promoter and gene body methylation better predicts gene expression than promoter methylation alone including genes involved in the neuroendocrine classification of SCLC and the expression of therapeutically relevant genes including MGMT, SLFN11, and DLL3. Importantly, for super-enhancer (SE) covered genes such as NEUROD1 or MYC, using H3K27ac and NEUROD1, ASCL1, and POU2F3 ChIP-seq data, we show that genic methylation is inversely proportional to expression, thus providing a new approach to identify potential SE regulated genes involved in SCLC pathogenesis. To advance SCLC transitional research, these data are integrated into our web portal (https://discover.nci.nih.gov/SclcCellMinerCDB/) for open and easy access to basic and clinical investigators.

Replication stress defines distinct molecular subtypes across cancers.

Endogenous replication stress is a major driver of genomic instability. Current assessments of replication stress are low throughput precluding its comprehensive assessment across tumors. Here we develop and validate a transcriptional profile of replication stress by leveraging established cellular characteristics that portend replication stress. The repstress gene signature defines a subset of tumors across lineages characterized by activated oncogenes, aneuploidy, extrachromosomal DNA amplification, immune evasion, high genomic instability, and poor survival, and importantly predicts response to agents targeting replication stress more robustly than previously reported transcriptomic measures of replication stress. Repstress score profiles the dual roles of replication stress during tumorigenesis and in established cancers and defines distinct molecular subtypes within cancers that may be more vulnerable to drugs targeting this dependency. Altogether, our study provides a molecular profile of replication stress, providing novel biological insights of the replication stress phenotype, with clinical implications.

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.

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.

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.

Exploring the Significance of the Exon 4-Skipping Isoform of the ZNF217 Oncogene in Breast Cancer.

Oncogene alternative splicing events can create distinct functional transcripts that offer new candidate prognostic biomarkers for breast cancer. ZNF217 is a well-established oncogene but its exon 4-skipping isoform (ZNF217-ΔE4) has never been investigated in terms of clinical or biological relevance. Using in silico RNA-seq and RT-qPCR analyses, we demonstrated for the first time the existence of ZNF217-ΔE4 transcripts in primary breast tumors, and a positive correlation between ZNF217-ΔE4 mRNA levels and those of the wild-type oncogene (ZNF217-WT). A pilot retrospective analysis revealed that, in the Luminal subclass, the combination of the two ZNF217 variants (the ZNF217-ΔE4-WT gene-expression signature) provided more information than the mRNA expression levels of each isoform alone. Ectopic overexpression of ZNF217-ΔE4 in breast cancer cells promoted an aggressive phenotype and an increase in ZNF217-WT expression levels that was inversely correlated with DNA methylation of the ZNF217 gene. This study provides new insights into the possible role of the ZNF217-ΔE4 splice variant in breast cancer and suggests a close interplay between the ZNF217-WT and ZNF217-ΔE4 isoforms. Our data suggest that a dual signature combining the expression levels of these two isoforms may serve as a novel prognostic biomarker allowing better stratification of breast cancers with good prognosis and aiding clinicians in therapeutic decisions.

Dynamics of replication origin over-activation.

Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.

Whole-exome sequencing reveals germline-mutated small cell lung cancer subtype with favorable response to DNA repair-targeted therapies.

Because tobacco is a potent carcinogen, secondary causes of lung cancer are often diminished in perceived importance. To assess the extent of inherited susceptibility to small cell lung cancer (SCLC), the most lethal type of lung cancer, we sequenced germline exomes of 87 patients (77 SCLC and 10 extrapulmonary small cell) and considered 607 genes, discovering 42 deleterious variants in 35 cancer-predisposition genes among 43.7% of patients. These findings were validated in an independent cohort of 79 patients with SCLC. Loss of heterozygosity was observed in 3 of 14 (21.4%) tumors. Identification of variants influenced medical management and family member testing in nine (10.3%) patients. Unselected patients with SCLC were more likely to carry germline RAD51 paralog D (RAD51D), checkpoint kinase 1 (CHEK1), breast cancer 2 (BRCA2), and mutY DNA glycosylase (MUTYH) pathogenic variants than healthy controls. Germline genotype was significantly associated with the likelihood of a first-degree relative with cancer or lung cancer (odds ratio: 1.82, P = 0.008; and 2.60, P = 0.028), and longer recurrence-free survival after platinum-based chemotherapy (P = 0.002), independent of known prognostic factors. Treatment of a patient with relapsed SCLC and germline pathogenic mutation of BRCA1 interacting protein C-terminal helicase 1 (BRIP1), a homologous recombination-related gene, using agents synthetically lethal with homologous recombination deficiency, resulted in a notable disease response. This work demonstrates that SCLC, currently thought to result almost exclusively from tobacco exposure, may have an inherited predisposition and lays the groundwork for targeted therapies based on the genes involved.

Genome-Wide Analysis Unveils DNA Helicase RECQ1 as a Regulator of Estrogen Response Pathway in Breast Cancer Cells.

Susceptibility to breast cancer is significantly increased in individuals with germ line mutations in RECQ1 (also known as RECQL or RECQL1), a gene encoding a DNA helicase essential for genome maintenance. We previously reported that RECQ1 expression predicts clinical outcomes for sporadic breast cancer patients stratified by estrogen receptor (ER) status. Here, we utilized an unbiased integrative genomics approach to delineate a cross talk between RECQ1 and ERα, a known master regulatory transcription factor in breast cancer. We found that expression of ESR1, the gene encoding ERα, is directly activated by RECQ1. More than 35% of RECQ1 binding sites were cobound by ERα genome-wide. Mechanistically, RECQ1 cooperates with FOXA1, the pioneer transcription factor for ERα, to enhance chromatin accessibility at the ESR1 regulatory regions in a helicase activity-dependent manner. In clinical ERα-positive breast cancers treated with endocrine therapy, high RECQ1 and high FOXA1 coexpressing tumors were associated with better survival. Collectively, these results identify RECQ1 as a novel cofactor for ERα and uncover a previously unknown mechanism by which RECQ1 regulates disease-driving gene expression in ER-positive breast cancer cells.

PM4NGS, a project management framework for next-generation sequencing data analysis.

BACKGROUND: FAIR (Findability, Accessibility, Interoperability, and Reusability) next-generation sequencing (NGS) data analysis relies on complex computational biology workflows and pipelines to guarantee reproducibility, portability, and scalability. Moreover, workflow languages, managers, and container technologies have helped address the problem of data analysis pipeline execution across multiple platforms in scalable ways. FINDINGS: Here, we present a project management framework for NGS data analysis called PM4NGS. This framework is composed of an automatic creation of a standard organizational structure of directories and files, bioinformatics tool management using Docker or Bioconda, and data analysis pipelines in CWL format. Pre-configured Jupyter notebooks with minimum Python code are included in PM4NGS to produce a project report and publication-ready figures. We present 3 pipelines for demonstration purposes including the analysis of RNA-Seq, ChIP-Seq, and ChIP-exo datasets. CONCLUSIONS: PM4NGS is an open source framework that creates a standard organizational structure for NGS data analysis projects. PM4NGS is easy to install, configure, and use by non-bioinformaticians on personal computers and laptops. It permits execution of the NGS data analysis on Windows 10 with the Windows Subsystem for Linux feature activated. The framework aims to reduce the gap between researcher in experimental laboratories producing NGS data and workflows for data analysis. PM4NGS documentation can be accessed at https://pm4ngs.readthedocs.io/.

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.

DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks.

The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a "self-trapping" mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2.