Unveiling the Dynamics behind Glioblastoma Multiforme Single-Cell Data Heterogeneity.

Glioblastoma Multiforme is a brain tumor distinguished by its aggressiveness. We suggested that this aggressiveness leads single-cell RNA-sequence data (scRNA-seq) to span a representative portion of the cancer attractors domain. This conjecture allowed us to interpret the scRNA-seq heterogeneity as reflecting a representative trajectory within the attractor's domain. We considered factors such as genomic instability to characterize the cancer dynamics through stochastic fixed points. The fixed points were derived from centroids obtained through various clustering methods to verify our method sensitivity. This methodological foundation is based upon sample and time average equivalence, assigning an interpretative value to the data cluster centroids and supporting parameters estimation. We used stochastic simulations to reproduce the dynamics, and our results showed an alignment between experimental and simulated dataset centroids. We also computed the Waddington landscape, which provided a visual framework for validating the centroids and standard deviations as characterizations of cancer attractors. Additionally, we examined the stability and transitions between attractors and revealed a potential interplay between subtypes. These transitions might be related to cancer recurrence and progression, connecting the molecular mechanisms of cancer heterogeneity with statistical properties of gene expression dynamics. Our work advances the modeling of gene expression dynamics and paves the way for personalized therapeutic interventions.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Loss of cytidine deaminase expression as a potential attempt to counteract the process of carcinogenesis by reducing basal PARP-1 activity and increasing tau levels.

Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that catalyzes the hydrolytic deamination of free cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Our team discovered that CDA deficiency is associated with several aspects of genetic instability, such as increased sister chromatid exchange and ultrafine anaphase bridge frequencies. Based on these results, we sought (1) to determine how CDA deficiency contributes to genetic instability, (2) to explore the possible relationships between CDA deficiency and carcinogenesis, and (3) to develop a new anticancer treatment targeting CDA-deficient tumors. This review summarizes our major findings indicating that CDA deficiency is associated with a genetic instability that does not confer an increased cancer risk. In light of our results and published data, I propose a novel hypothesis that loss of CDA, by reducing basal PARP-1 activity and increasing Tau levels, may reflect an attempt to prevent, slow or reverse the process of carcinogenesis.

Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.

Pivotal functions and impact of long con-coding RNAs on cellular processes and genome integrity.

Recent advances in uncovering the mysteries of the human genome suggest that long non-coding RNAs (lncRNAs) are important regulatory components. Although lncRNAs are known to affect gene transcription, their mechanisms and biological implications are still unclear. Experimental research has shown that lncRNA synthesis, subcellular localization, and interactions with macromolecules like DNA, other RNAs, or proteins can all have an impact on gene expression in various biological processes. In this review, we highlight and discuss the major mechanisms through which lncRNAs function as master regulators of the human genome. Specifically, the objective of our review is to examine how lncRNAs regulate different processes like cell division, cell cycle, and immune responses, and unravel their roles in maintaining genomic architecture and integrity.

DDX21: The link between m6A and R-loops.

In this issue of Molecular Cell, Hao et al.1 demonstrate that the RNA helicase DDX21 recruits the m6A methyltransferase complex to R-loops, ensuring proper transcription termination and genome stability.

DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease.

Crosstalk between DNA Damage Repair and Metabolic Regulation in Hematopoietic Stem Cells.

Self-renewal and differentiation are two characteristics of hematopoietic stem cells (HSCs). Under steady physiological conditions, most primitive HSCs remain quiescent in the bone marrow (BM). They respond to different stimuli to refresh the blood system. The transition from quiescence to activation is accompanied by major changes in metabolism, a fundamental cellular process in living organisms that produces or consumes energy. Cellular metabolism is now considered to be a key regulator of HSC maintenance. Interestingly, HSCs possess a distinct metabolic profile with a preference for glycolysis rather than oxidative phosphorylation (OXPHOS) for energy production. Byproducts from the cellular metabolism can also damage DNA. To counteract such insults, mammalian cells have evolved a complex and efficient DNA damage repair (DDR) system to eliminate various DNA lesions and guard genomic stability. Given the enormous regenerative potential coupled with the lifetime persistence of HSCs, tight control of HSC genome stability is essential. The intersection of DDR and the HSC metabolism has recently emerged as an area of intense research interest, unraveling the profound connections between genomic stability and cellular energetics. In this brief review, we delve into the interplay between DDR deficiency and the metabolic reprogramming of HSCs, shedding light on the dynamic relationship that governs the fate and functionality of these remarkable stem cells. Understanding the crosstalk between DDR and the cellular metabolism will open a new avenue of research designed to target these interacting pathways for improving HSC function and treating hematologic disorders.

Full-spectral genome analysis of natural killer/T cell lymphoma highlights impacts of genome instability in driving its progression.

BACKGROUND: Natural killer/T cell lymphoma (NKTCL) is a clinically and genetically heterogeneous disease with poor prognosis. Genome sequencing and mutation characterization provides a powerful approach for patient stratification, treatment target discovery, and etiology identification. However, previous studies mostly concentrated on base-level mutations in primary NKTCL, whereas the large-scale genomic alterations in NKTCL and the mutational landscapes in relapsed/refractory NKTCL remain largely unexplored. METHODS: Here, we assembled whole-genome sequencing and whole-exome sequencing data from 163 patients with primary or relapsed/refractory NKTCL and compared their somatic mutational landscapes at both nucleotide and structure levels. RESULTS: Our study not only confirmed previously reported common NKTCL mutational targets like STAT3, TP53, and DDX3X but also unveiled several novel high-frequency mutational targets such as PRDM9, DST, and RBMX. In terms of the overall mutational landscape, we observed striking differences between primary and relapsed/refractory NKTCL patient groups, with the latter exhibits higher levels of tumor mutation burden, copy number variants (CNVs), and structural variants (SVs), indicating a strong signal of genomic instability. Complex structural rearrangements such as chromothripsis and focal amplification are also significantly enriched in relapsed/refractory NKTCL patients, exerting a substantial impact on prognosis. Accordingly, we devised a novel molecular subtyping system (i.e., C0-C4) with distinct prognosis by integrating potential driver mutations at both nucleotide and structural levels, which further provides an informative guidance for novel treatments that target these specific driver mutations and genome instability as a whole. CONCLUSIONS: The striking differences underlying the mutational landscapes between the primary and relapsed/refractory NKTCL patients highlight the importance of genomic instability in driving the progression of NKTCL. Our newly proposed molecular subtyping system is valuable in assisting patient stratification and novel treatment design towards a better prognosis in the age of precision medicine.

Surviving without BRCA2: MLH1 gets R-looped in to curtail genomic instability.

While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.

Prediction of deleterious non-synonymous SNPs of human MDC1 gene: an in silico approach.

MDC1 (Mediator of DNA damage Checkpoint protein 1) functions to facilitate the localization of numerous DNA damage response (DDR) components to DNA double-strand break sites. MDC1 is an integral component in preserving genomic stability and appropriate DDR regulation. There haven't been systematic investigations of MDC1 mutations that induce cancer and genomic instability. Variations in nsSNPs have the potential to modify the protein chemistry and their function. Describing functional SNPs in disease-associated genes presents a significant conundrum for investigators, it is possible to assess potential functional SNPs before conducting larger population examinations. Multiple sequences and structure-based bioinformatics strategies were implemented in the current in-silico investigation to discern potential nsSNPs of the MDC1 genes. The nsSNPs were identified with SIFT, SNAP2, Align GVGD, PolyPhen-2, and PANTHER, and their stability was determined with MUpro. The conservation, solvent accessibility, and structural effects of the mutations were identified with ConSurf, NetSurfP-2.0, and SAAFEC-SEQ respectively. Cancer-related analysis of the nsSNPs was conducted using cBioPortal and TCGA web servers. The present study appraised five nsSNPs (P1426T, P69S, P194R, P203L, and H131Y) as probably mutilating due to their existence in highly conserved regions and propensity to deplete protein stability. The nsSNPs P194R, P203L, and H131Y were concluded as deleterious and possibly damaging from the 5 prediction tools. The functional nsSNP P194R mutation is associated with skin cutaneous melanoma while no significant records were found for other nsSNPs. The present study concludes that the highly deleterious P194R mutations can potentially induce genomic instability and contribute to various cancers' pathogenesis. Developing drugs targeting these mutations can undoubtedly be advantageous in large population-based studies, particularly in the development of precision medicine.

TRIM33 loss in multiple myeloma is associated with genomic instability and sensitivity to PARP inhibitors.

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Living Beyond Restriction: LBR promotes cellular immortalization by suppressing genomic instability and senescence.

Cellular immortalization is a complex process that requires multiple genetic alterations to overcome restricting barriers, including senescence. Not surprisingly, many of these alterations are associated with cancer; two tumor suppressor pathways, the cellular tumor antigen p53 and p16-Retinoblastoma (RB) pathways, are the best-characterized examples, but their mutations alone are known to be insufficient to drive full immortalization. En et al. identified a role for the lamin B receptor (LBR) in promoting cellular proliferation and immortalization in p53- and RB-deficient cells by maintaining their genome integrity and suppressing senescence. Thus, modulation of LBR could be exploited to treat cancer and potentially also to promote cell rejuvenation.

R-loop and diseases: the cell cycle matters.

The cell cycle is a crucial biological process that is involved in cell growth, development, and reproduction. It can be divided into G1, S, G2, and M phases, and each period is closely regulated to ensure the production of two similar daughter cells with the same genetic material. However, many obstacles influence the cell cycle, including the R-loop that is formed throughout this process. R-loop is a triple-stranded structure, composed of an RNA: DNA hybrid and a single DNA strand, which is ubiquitous in organisms from bacteria to mammals. The existence of the R-loop has important significance for the regulation of various physiological processes. However, aberrant accumulation of R-loop due to its limited resolving ability will be detrimental for cells. For example, DNA damage and genomic instability, caused by the R-loop, can activate checkpoints in the cell cycle, which in turn induce cell cycle arrest and cell death. At present, a growing number of factors have been proven to prevent or eliminate the accumulation of R-loop thereby avoiding DNA damage and mutations. Therefore, we need to gain detailed insight into the R-loop resolution factors at different stages of the cell cycle. In this review, we review the current knowledge of factors that play a role in resolving the R-loop at different stages of the cell cycle, as well as how mutations of these factors lead to the onset and progression of diseases.

Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

RAD52-dependent mitotic DNA synthesis is required for genome stability in Cyclin E1-overexpressing cells.

Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.

Non-canonical role for the BAF complex subunit DPF3 in mitosis and ciliogenesis.

DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis.

The nucleolus: Coordinating stress response and genomic stability.

The perception that the nucleoli are merely the organelles where ribosome biogenesis occurs is challenged. Only around 30 % of nucleolar proteins are solely involved in producing ribosomes. Instead, the nucleolus plays a critical role in controlling protein trafficking during stress and, according to its dynamic nature, undergoes continuous protein exchange with nucleoplasm under various cellular stressors. Hence, the concept of nucleolar stress has evolved as cellular insults that disrupt the structure and function of the nucleolus. Considering the emerging role of this organelle in DNA repair and the fact that rDNAs are the most fragile genomic loci, therapies targeting the nucleoli are increasingly being developed. Besides, drugs that target ribosome synthesis and induce nucleolar stress can be used in cancer therapy. In contrast, agents that regulate nucleolar activity may be a potential treatment for neurodegeneration caused by abnormal protein accumulation in the nucleolus. Here, I explore the roles of nucleoli beyond their ribosomal functions, highlighting the factors triggering nucleolar stress and their impact on genomic stability.

Characterization of the prognostic, diagnostic, and immunological roles of DSCC1 and its genomic alteration and instability in human cancers.

DNA replication and sister chromatid cohesion 1 (DSCC1) exerts various functions including sister chromatid cohesion. DSCC1 overexpression plays an important role in cancer development, such as in colorectal, breast, and hepatocellular cancers. The specific role of DSCC1 in tumor progression remains largely unknown, necessitating a pan-cancer investigation to understand the potential function of DSCC1 in various cancers. In this study, we obtained data on physiological conditions, transcriptional expression, survival prognosis, genomic alteration, genomic instability, enriched pathways, immune infiltration, and immunotherapy from The Cancer Genome Atlas, The Genotype-Tissue Expression, cBioPortal, and other publicly available databases to systematically characterize the oncogenic and immunological roles of DSCC1 in 33 different cancers. We found that DSCC1 expression was upregulated at both mRNA and protein levels in various cancers. Additionally, DSCC1 expression was associated with higher tumor stage and grade in specific cancers. DSCC1 was a potential pan-cancer prognostic biomarker for its close association with patient prognosis and a diagnostic biomarker for its high predictive value in distinguishing tumor tissues from normal tissues. DSCC1 was universally amplified across different cancers and tightly associated with genomic instability. Moreover, DSCC1 had a close relationship with tumor immune cell infiltration; thus, it could be used as a potential biomarker for predicting the response and survival of patients with cancer who receive immune checkpoint blockade treatment. To sum up, our study revealed that DSCC1 is a promising target for tumor therapy.