ABSTRACT
One possible carbon dioxide sequestration strategy is via the carbonation of dissolved Mg2+ obtained through olivine ((Mg,Fe)2SiO4) dissolution. However, silica is also produced during the breakdown of olivine. This component may have a detrimental effect on the yield of Mg-carbonate as Mg2+ incorporation into complex Mg silicate phases would limit CO2 uptake by this system. Yet this potential competition is currently not considered. Here, we use crystal growth experiments at temperatures applicable for potential coastal applications to test the effect of silica on the formation of the hydrated Mg-carbonate phase nesquehonite (MgCO3·3H2O). Solution chemistry analysis coupled with phase identification demonstrates that the presence of silica in the solution can actually assist the formation of nesquehonite and increase its yield by as much as 60 times. Our findings suggest that the presence of silica changes interfacial stabilities, lowering the energetic barrier for nesquehonite nucleation. In addition, in situ attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) transformation experiments demonstrated that nesquehonite precipitating in a solution containing a high concentration of dissolved silica exhibits enhanced stability against its transformation into hydromagnesite. These findings will help to better constrain what we expect for applications of olivine during carbon remediation strategies as well as assist yields for industrial applications that use Mg-based cement as building materials to facilitate a CO2-neutral or negative footprint.
Subject(s)
Magnesium , Silicon Dioxide , Magnesium/chemistry , Carbonates , Carbon Dioxide/chemistryABSTRACT
While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.
Subject(s)
Brain Neoplasms/genetics , DNA Replication/genetics , Genomic Instability , Glioma/genetics , Histones/physiology , Brain Neoplasms/pathology , Child , Gene Expression Regulation, Neoplastic , Glioma/pathology , Humans , Mitosis/geneticsABSTRACT
Tumours with mutations in the BRCA1/BRCA2 genes have impaired double-stranded DNA break repair, compromised replication fork protection and increased sensitivity to replication blocking agents, a phenotype collectively known as 'BRCAness'. Tumours with a BRCAness phenotype become dependent on alternative repair pathways that are error-prone and introduce specific patterns of somatic mutations across the genome. The increasing availability of next-generation sequencing data of tumour samples has enabled identification of distinct mutational signatures associated with BRCAness. These signatures reveal that alternative repair pathways, including Polymerase θ-mediated alternative end-joining and RAD52-mediated single strand annealing are active in BRCA1/2-deficient tumours, pointing towards potential therapeutic targets in these tumours. Additionally, insight into the mutations and consequences of unrepaired DNA lesions may also aid in the identification of BRCA-like tumours lacking BRCA1/BRCA2 gene inactivation. This is clinically relevant, as these tumours respond favourably to treatment with DNA-damaging agents, including PARP inhibitors or cisplatin, which have been successfully used to treat patients with BRCA1/2-defective tumours. In this review, we aim to provide insight in the origins of the mutational landscape associated with BRCAness by exploring the molecular biology of alternative DNA repair pathways, which may represent actionable therapeutic targets in in these cells.
Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA Breaks, Double-Stranded , DNA Repair/genetics , Neoplasms/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cisplatin/pharmacology , Cisplatin/therapeutic use , DNA Breaks, Double-Stranded/drug effects , DNA Repair/drug effects , Humans , Mutation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic useABSTRACT
Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.
Subject(s)
Neoplasms , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Cyclin E/genetics , Cyclin E/metabolism , Genomic Instability , Protein Phosphatase 2C/genetics , Protein Phosphatase 2C/metabolismABSTRACT
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.
Subject(s)
Cyclin E , Genomic Instability , Mitosis , Oncogene Proteins , Rad52 DNA Repair and Recombination Protein , Humans , Cyclin E/metabolism , Cyclin E/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Rad52 DNA Repair and Recombination Protein/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , DNA Replication , Cell Line, Tumor , DNA Damage , DNA/metabolism , DNA/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathologyABSTRACT
Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.
Subject(s)
Mitosis , Proteins , Proteins/genetics , Adenosine Triphosphatases/metabolism , DNA , Chromatids/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , DNA Replication/genetics , DNA DamageABSTRACT
Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors , Sister Chromatid Exchange , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Chromosome Fragile Sites , Homologous Recombination/genetics , DNAABSTRACT
Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.
Subject(s)
DNA Damage , DNA Repair , Genomic Instability , Peptide Elongation Factor 1/metabolism , Transcription Elongation, Genetic , CRISPR-Cas Systems , Carrier Proteins/genetics , Carrier Proteins/metabolism , Evolution, Molecular , HCT116 Cells , Humans , Peptide Elongation Factor 1/genetics , RNA Polymerase II/metabolism , Transcription Factor TFIIH/genetics , Transcription Factor TFIIH/metabolism , UbiquitinationABSTRACT
Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.
ABSTRACT
Poly(ADP-ribose) polymerase (PARP) inhibitors are selectively cytotoxic in cancer cells with defects in homologous recombination (HR) (e.g., due to BRCA1/2 mutations). However, not all HR-deficient tumors efficiently respond to PARP inhibition and often acquire resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR-deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2-depleted and Brca2 knockout cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome-wide single-cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib-treated BRCA2-depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING-associated inflammatory response in BRCA2-deficient cells. ATR inhibition further increased the numbers of cGAS-positive micronuclei and the extent of cytokine production in olaparib-treated BRCA2-deficient cancer cells. Altogether, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR-deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.
Subject(s)
Apoptosis/drug effects , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , BRCA2 Protein/deficiency , Genomic Instability/drug effects , Inflammation/pathology , Mitosis , Phthalazines/pharmacology , Piperazines/pharmacology , Signal Transduction , Apoptosis/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA2 Protein/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/metabolism , DNA Damage , G2 Phase Cell Cycle Checkpoints/drug effects , HeLa Cells , Humans , Micronuclei, Chromosome-Defective/drug effects , Mitosis/drug effects , Nucleotidyltransferases/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Signal Transduction/drug effectsABSTRACT
Triple-negative breast cancers (TNBCs) display great diversity in cisplatin sensitivity that cannot be explained solely by cancer-associated DNA repair defects. Differential activation of the DNA damage response (DDR) to cisplatin has been proposed to underlie the observed differential sensitivity, but it has not been investigated systematically. Systems-level analysis-using quantitative time-resolved signaling data and phenotypic responses, in combination with mathematical modeling-identifies that the activation status of cell-cycle checkpoints determines cisplatin sensitivity in TNBC cell lines. Specifically, inactivation of the cell-cycle checkpoint regulator MK2 or G3BP2 sensitizes cisplatin-resistant TNBC cell lines to cisplatin. Dynamic signaling data of five cell cycle-related signals predicts cisplatin sensitivity of TNBC cell lines. We provide a time-resolved map of cisplatin-induced signaling that uncovers determinants of chemo-sensitivity, underscores the impact of cell-cycle checkpoints on cisplatin sensitivity, and offers starting points to optimize treatment efficacy.
Subject(s)
Drug Resistance, Neoplasm , Models, Theoretical , Triple Negative Breast Neoplasms/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Antineoplastic Agents/pharmacology , Cell Cycle , Cell Line, Tumor , Cisplatin/pharmacology , DNA Damage , Female , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction/drug effectsABSTRACT
Genomic instability is a hallmark feature of cancer cells, and can be caused by defective DNA repair, for instance due to inactivation of BRCA2. Paradoxically, loss of Brca2 in mice results in embryonic lethality, whereas cancer cells can tolerate BRCA2 loss. This holds true for multiple DNA repair genes, and suggests that cancer cells are molecularly "rewired" to cope with defective DNA repair and the resulting high levels of genomic instability. In this study, we aim to identify genes that genomically unstable cancer cells rely on for their survival. Using functional genomic mRNA (FGmRNA) profiling, 16,172 cancer samples were previously ranked based on their degree of genomic instability. We analyzed the top 250 genes that showed a positive correlation between FGmRNA levels and the degree of genomic instability, in a co-functionality network. Within this co-functionality network, a strong cluster of 11 cell cycle-related genes was identified, including TPX2. We then assessed the dependency on these 11 genes in the context of survival of genomically unstable cancer cells, induced by BRCA2 inactivation. Depletion of TPX2 or its associated kinase Aurora-A preferentially reduced cell viability in a panel of BRCA2-deficient cancer cells. In line with these findings, BRCA2-depleted and BRCA2-mutant human cell lines, or tumor cell lines derived from Brca2-/-;p53-/- mice showed increased sensitivity to the Aurora-A kinase inhibitor alisertib, with delayed mitotic progression and frequent mitotic failure. Our findings reveal that BRCA2-deficient cancer cells show enhanced sensitivity to inactivation of TPX2 or its partner Aurora-A, which points at an actionable dependency of genomically unstable cancers.