H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours.

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

Digital Twins in Agriculture and Forestry: A Review.

Digital twins aim to optimize practices implemented in various sectors by bridging the gap between the physical and digital worlds. Focusing on open-field agriculture, livestock farming, and forestry and reviewing the current applications in these domains, this paper reveals the multifaceted roles of digital twins. Diverse key aspects are examined, including digital twin integration and maturity level, means of data acquisition, technological capabilities, and commonly used input and output features. Through the prism of four primary research questions, the state of the art of digital twins, the extent of their achieved integration, and an overview of the critical issues and potential advancements are provided in the landscape of the sectors under consideration. The paper concludes that in spite of the remarkable progress, there is a long way towards achieving full digital twin. Challenges still persist, while the key factor seems to be the integration of expert knowledge from different stakeholders. In light of the constraints identified in the review analysis, a new sector-specific definition for digital twins is also suggested to align with the distinctive characteristics of intricate biotic and abiotic systems. This research is anticipated to serve as a useful reference for stakeholders, enhancing awareness of the considerable benefits associated with digital twins and promoting a more systematic and comprehensive exploration of this transformative topic.

PALB2-mutated human mammary cells display a broad spectrum of morphological and functional abnormalities induced by increased TGFß signaling.

Heterozygous mutations in any of three major genes, BRCA1, BRCA2 and PALB2, are associated with high-risk hereditary breast cancer susceptibility frequently seen as familial disease clustering. PALB2 is a key interaction partner and regulator of several vital cellular activities of BRCA1 and BRCA2, and is thus required for DNA damage repair and alleviation of replicative and oxidative stress. Little is however known about how PALB2-deficiency affects cell function beyond that, especially in the three-dimensional setting, and also about its role during early steps of malignancy development. To answer these questions, we have generated biologically relevant MCF10A mammary epithelial cell lines with mutations that are comparable to certain clinically important PALB2 defects. We show in a non-cancerous background how both mono- and biallelically PALB2-mutated cells exhibit gross spontaneous DNA damage and mitotic aberrations. Furthermore, PALB2-deficiency disturbs three-dimensional spheroid morphology, increases the migrational capacity and invasiveness of the cells, and broadly alters their transcriptome profiles. TGFß signaling and KRT14 expression are enhanced in PALB2-mutated cells and their inhibition and knock down, respectively, lead to partial restoration of cell functions. KRT14-positive cells are also more abundant with DNA damage than KRT14-negative cells. The obtained results indicate comprehensive cellular changes upon PALB2 mutations, even in the presence of half dosage of wild type PALB2 and demonstrate how PALB2 mutations may predispose their carriers to malignancy.

ADAM12 expression is upregulated in cancer cells upon radiation and constitutes a prognostic factor in rectal cancer patients following radiotherapy.

Radiotherapy is one of the most common cancer treatments, yet, some patients require high doses to respond. Therefore, the development of new strategies leans toward personalizing therapy to avoid unnecessary burden on cancer patients. This approach prevents the administration of ineffective treatments or uses combination strategies to increase the sensitivity of cancer cells. ADAM12 has been shown to be upregulated in many cancers and correlate with poor survival and chemoresistance, thus making it a potential candidate responsible for radioresistance. Here, we show that ADAM12 expression is upregulated in response to irradiation in both mouse and human cancer cells in vitro, as well as in tumor tissues from rectal cancer patients. Interestingly, the expression of ADAM12 following radiotherapy correlates with the initial disease stage and predicts the response of rectal cancer patients to the treatment. While we found no cell-autonomous effects of ADAM12 on the response of colon cancer cells to irradiation in vitro, depletion of ADAM12 expression markedly reduced the tumor growth of irradiated cancer cells when subcutaneously transplanted in syngeneic mice. Interestingly, loss of cancer cell-derived ADAM12 expression increased the number of CD31+FAP- cells in murine tumors. Moreover, conditioned medium from ADAM12-/- colon cancer cells led to increased tube formation when added to endothelial cell cultures. Thus, it is tempting to speculate that altered tumor vascularity may be implicated in the observed effect of ADAM12 on response to radiotherapy in rectal cancer. We conclude that ADAM12 represents a promising prognostic factor for stratification of rectal cancer patients receiving radiotherapy and suggest that targeting ADAM12 in combination with radiotherapy could potentially improve the treatment response.

Synthetic lethal interaction between WEE1 and PKMYT1 is a target for multiple low-dose treatment of high-grade serous ovarian carcinoma.

Ovarian cancer is driven by genetic alterations that necessitate protective DNA damage and replication stress responses through cell cycle control and genome maintenance. This creates specific vulnerabilities that may be exploited therapeutically. WEE1 kinase is a key cell cycle control kinase, and it has emerged as a promising cancer therapy target. However, adverse effects have limited its clinical progress, especially when tested in combination with chemotherapies. A strong genetic interaction between WEE1 and PKMYT1 led us to hypothesize that a multiple low-dose approach utilizing joint WEE1 and PKMYT1 inhibition would allow exploitation of the synthetic lethality. We found that the combination of WEE1 and PKMYT1 inhibition exhibited synergistic effects in eradicating ovarian cancer cells and organoid models at a low dose. The WEE1 and PKMYT1 inhibition synergistically promoted CDK activation. Furthermore, the combined treatment exacerbated DNA replication stress and replication catastrophe, leading to increase of the genomic instability and inflammatory STAT1 signalling activation. These findings suggest a new multiple low-dose approach to harness the potency of WEE1 inhibition through the synthetic lethal interaction with PKMYT1 that may contribute to the development of new treatments for ovarian cancer.

Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

Self-inflicted DNA breaks in cell differentiation and cancer.

Self-inflicted DNA strand breaks are canonically linked with cell death pathways and the establishment of genetic diversity in immune and germline cells. Moreover, this form of DNA damage is an established source of genome instability in cancer development. However, recent studies indicate that nonlethal self-inflicted DNA strand breaks play an indispensable but underappreciated role in a variety of cell processes, including differentiation and cancer therapy responses. Mechanistically, these physiological DNA breaks originate from the activation of nucleases, which are best characterized for inducing DNA fragmentation in apoptotic cell death. In this review, we outline the emerging biology of one critical nuclease, caspase-activated DNase (CAD), and how directed activation or deployment of this enzyme can lead to divergent cell fate outcomes.

Temporal phosphoproteomics reveals WEE1-dependent control of 53BP1 pathway.

Wee1-like protein kinase (WEE1) restrains activities of cyclin-dependent kinases (CDKs) in S and G2 phase. Inhibition of WEE1 evokes drastic increase in CDK activity, which perturbs replication dynamics and compromises cell cycle checkpoints. Notably, WEE1 inhibitors such as adavosertib are tested in cancer treatment trials; however, WEE1-regulated phosphoproteomes and their dynamics have not been systematically investigated. In this study, we identified acute time-resolved alterations in the cellular phosphoproteome following WEE1 inhibition with adavosertib. These treatments acutely elevated CDK activities with distinct phosphorylation dynamics revealing more than 600 potential uncharacterized CDK sites. Moreover, we identified a major role for WEE1 in controlling CDK-dependent phosphorylation of multiple clustered sites in the key DNA repair factors MDC1, 53BP1, and RIF1. Functional analysis revealed that WEE1 fine-tunes CDK activities to permit recruitment of 53BP1 to chromatin. Thus, our findings uncover WEE1-controlled targets and pathways with translational potential for the clinical application of WEE1 inhibitors.

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation.

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

Multiparametric and accurate functional analysis of genetic sequence variants using CRISPR-Select.

Determining the functional role of thousands of genetic sequence variants (mutations) associated with genetic diseases is a major challenge. Here we present clustered regularly interspaced short palindromic repeat (CRISPR)-SelectTIME, CRISPR-SelectSPACE and CRISPR-SelectSTATE, a set of flexible knock-in assays that introduce a genetic variant in a cell population and track its absolute frequencies relative to an internal, neutral control mutation as a function of time, space or a cell state measurable by flow cytometry. Phenotypically, CRISPR-Select can thereby determine, for example, pathogenicity, drug responsiveness/resistance or in vivo tumor promotion by a specific variant. Mechanistically, CRISPR-Select can dissect how the variant elicits the phenotype by causally linking the variant to motility/invasiveness or any cell state or biochemical process with a flow cytometry marker. The method is applicable to organoids, nontransformed or cancer cell lines. It is accurate, quantitative, fast and simple and works in single-well or 96-well higher throughput format. CRISPR-Select provides a versatile functional variant assay for research, diagnostics and drug development for genetic disorders.

Lamin A/C impairments cause mitochondrial dysfunction by attenuating PGC1α and the NAMPT-NAD+ pathway.

Mutations in the lamin A/C gene (LMNA) cause laminopathies such as the premature aging Hutchinson Gilford progeria syndrome (HGPS) and altered lamin A/C levels are found in diverse malignancies. The underlying lamin-associated mechanisms remain poorly understood. Here we report that lamin A/C-null mouse embryo fibroblasts (Lmna-/- MEFs) and human progerin-expressing HGPS fibroblasts both display reduced NAD+ levels, unstable mitochondrial DNA and attenuated bioenergetics. This mitochondrial dysfunction is associated with reduced chromatin recruitment (Lmna-/- MEFs) or low levels (HGPS) of PGC1α, the key transcription factor for mitochondrial homeostasis. Lmna-/- MEFs showed reduced expression of the NAD+-biosynthesis enzyme NAMPT and attenuated activity of the NAD+-dependent deacetylase SIRT1. We find high PARylation in lamin A/C-aberrant cells, further decreasing the NAD+ pool and consistent with impaired DNA base excision repair in both cell models, a condition that fuels DNA damage-induced PARylation under oxidative stress. Further, ATAC-sequencing revealed a substantially altered chromatin landscape in Lmna-/- MEFs, including aberrantly reduced accessibility at the Nampt gene promoter. Thus, we identified a new role of lamin A/C as a key modulator of mitochondrial function through impairments of PGC1α and the NAMPT-NAD+ pathway, with broader implications for the aging process.

Cancer cells use self-inflicted DNA breaks to evade growth limits imposed by genotoxic stress.

Genotoxic therapy such as radiation serves as a frontline cancer treatment, yet acquired resistance that leads to tumor reoccurrence is frequent. We found that cancer cells maintain viability during irradiation by reversibly increasing genome-wide DNA breaks, thereby limiting premature mitotic progression. We identify caspase-activated DNase (CAD) as the nuclease inflicting these de novo DNA lesions at defined loci, which are in proximity to chromatin-modifying CCCTC-binding factor (CTCF) sites. CAD nuclease activity is governed through phosphorylation by DNA damage response kinases, independent of caspase activity. In turn, loss of CAD activity impairs cell fate decisions, rendering cancer cells vulnerable to radiation-induced DNA double-strand breaks. Our observations highlight a cancer-selective survival adaptation, whereby tumor cells deploy regulated DNA breaks to delimit the detrimental effects of therapy-evoked DNA damage.

Convulsive Status Epilepticus Induced by Electroconvulsive Therapy in a Patient with Major Depression.

Electroconvulsive therapy (ECT) is a well-known, safe, and efficient treatment for a variety of psychiatric diseases. We present here an unusual case of a 34-year-old patient with major depression, who developed convulsive status epilepticus persistent for eight days in connection to her first ECT-a very uncommon but serious complication. The patient was, prior to ECT treatment, treated with lithium carbonate and clomipramine for her depression. Six years prior to the ECT, the patient had experienced a convulsive syncope resulting in traumatic subarachnoid haemorrhage. This case emphasizes the importance of medical recording to detect possible risk factors when considering ECT treatment.

WEE1 kinase protects the stability of stalled DNA replication forks by limiting CDK2 activity.

Cellular feedback systems ensure genome maintenance during DNA replication. When replication forks stall, newly replicated DNA is protected by pathways that limit excessive DNA nuclease attacks. Here we show that WEE1 activity guards against nascent DNA degradation at stalled forks. Furthermore, we identify WEE1-dependent suppression of cyclin-dependent kinase 2 (CDK2) as a major activity counteracting fork degradation. We establish DNA2 as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells. In addition, WEE1 appears to be unique among CDK activity suppressors in S phase because neither CHK1 nor p21 promote fork protection as WEE1 does. Our results identify a key role of WEE1 in protecting stalled forks, which is separate from its established role in safeguarding DNA replication initiation. Our findings highlight how WEE1 inhibition evokes massive genome challenges during DNA replication, and this knowledge may improve therapeutic strategies to specifically eradicate cancer cells that frequently harbor elevated DNA replication stress.

A catalog of curated breast cancer genes.

PURPOSE: Decades of research have identified multiple genetic variants associated with breast cancer etiology. However, there is no database that archives breast cancer genes and variants responsible for predisposition. We set out to build a dynamic repository of curated breast cancer genes. METHODS: A comprehensive literature search was performed in PubMed and Google Scholar, followed by data extraction and harmonization for downstream analysis. RESULTS: Using a subset of 345 studies, we cataloged 652 breast cancer-associated loci across the genome. A majority of these were present in the non-coding region (i.e., intergenic (101) and intronic (345)), whereas only 158 were located within an exon. Using the odds ratio, we identified 429 loci to increase the disease risk and 198 to confer protection against breast cancer, whereas 25 were identified to both increase disease risk and confer protection against breast cancer. Chromosomal ideogram analysis indicated that chromosomes 17 and 19 have the highest density of breast cancer loci. We manually annotated and collated breast cancer genes in which a previous association between rare-monogenic variant and breast cancer has been documented. Finally, network and functional enrichment analysis revealed that steroid metabolism and DNA repair pathways were predominant among breast cancer genes and variants. CONCLUSIONS: We have built an online interactive catalog of curated breast cancer genes ( https://cbcg.dk ). This will expedite clinical diagnostics and support the ongoing efforts in managing breast cancer etiology. Moreover, the database will serve as an essential repository when designing new breast cancer multigene panels.

Histone H4 lysine 20 mono-methylation directly facilitates chromatin openness and promotes transcription of housekeeping genes.

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.

Orchard Mapping with Deep Learning Semantic Segmentation.

This study aimed to propose an approach for orchard trees segmentation using aerial images based on a deep learning convolutional neural network variant, namely the U-net network. The purpose was the automated detection and localization of the canopy of orchard trees under various conditions (i.e., different seasons, different tree ages, different levels of weed coverage). The implemented dataset was composed of images from three different walnut orchards. The achieved variability of the dataset resulted in obtaining images that fell under seven different use cases. The best-trained model achieved 91%, 90%, and 87% accuracy for training, validation, and testing, respectively. The trained model was also tested on never-before-seen orthomosaic images or orchards based on two methods (oversampling and undersampling) in order to tackle issues with out-of-the-field boundary transparent pixels from the image. Even though the training dataset did not contain orthomosaic images, it achieved performance levels that reached up to 99%, demonstrating the robustness of the proposed approach.

Photochemotherapy Induces Interferon Type III Expression via STING Pathway.

DNA-damaging cancer therapies induce interferon expression and stimulate the immune system, promoting therapy responses. The immune-activating STING (Stimulator of Interferon Genes) pathway is induced when DNA or double-stranded RNA (dsRNA) is detected in the cell cytoplasm, which can be caused by viral infection or by DNA damage following chemo- or radiotherapy. Here, we investigated the responses of cutaneous T-cell lymphoma (CTCL) cells to the clinically applied DNA crosslinking photochemotherapy (combination of 8-methoxypsoralen and UVA light; 8-MOP + UVA). We showed that this treatment evokes interferon expression and that the type III interferon IFNL1 is the major cytokine induced. IFNL1 upregulation is dependent on STING and on the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS). Furthermore, 8-MOP + UVA treatment induced the expression of genes in pathways involved in response to the tumor necrosis factor, innate immune system and acute inflammatory response. Notably, a subset of these genes was under control of the STING-IFNL1 pathway. In conclusion, our data connected DNA damage with immune system activation via the STING pathway and contributed to a better understanding of the effectiveness of photochemotherapy.

Germline RBBP8 variants associated with early-onset breast cancer compromise replication fork stability.

Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. One significant pathway that is disabled as a result is homologous recombination repair (HRR). With the aim of identifying new candidate genes, we examined early-onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene), which mediates HRR through the end resection of DNA double-strand breaks (DSBs). Notably, these patients exhibited a number of rare germline RBBP8 variants. Functional analysis revealed that these variants did not affect DNA DSB end resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to that of cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which, when compromised, may predispose to the development of early-onset breast cancer.