Maintenance Of Genome Integrity Research Articles

Relationship of telomere length in colorectal cancer patients with cancer phenotype and patient prognosis

BackgroundTelomeres, repetitive DNA capping ends of eukaryotic chromosomes, are important in the maintenance of genomic integrity. Perturbed telomeres are common features of many human malignancies, including colorectal cancer.MethodsTelomere length (TL), measured by a Monochrome Multiplex Real-Time qPCR, was investigated in tumour tissues, adjacent mucosa, and blood from patients with colorectal cancer with different clinicopathological features and its impact on patient survival. TL was also measured in a limited number of liver metastases, non-cancerous liver tissues or corresponding tissues from the same patients.ResultsTL in tumour tissues was shorter than in the adjacent mucosa (P < 0.0001). Shorter TL was observed in tumours with lower stage than in those with advanced stages (P = 0.001). TL was shorter in tumours at the proximal than at the distal sites of the colon (P < 0.0001). Shorter TL was also associated with microsatellite instability (P = 0.001) and mucinous tumour histology (P < 0.0001). Patients with a smaller TL ratio between tumour tissues and the adjacent mucosa were associated with increased overall survival (P = 0.022). Metastasised tumours had shorter telomeres than the adjacent non-cancerous liver tissues (P = 0.0005).ConclusionsOverall, the results demonstrate differences in TL between tumours and the adjacent mucosa, between tumours located at different sites and association with patient survival.

Functional interplay between Mediator and RNA polymerase II in Rad2/XPG loading to the chromatin.

Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases.

Abstract 5068: Genomic regulation of the cell cycle as a function of ARID1A occupancy in breast epithelial cells

Abstract The idea of chemoprevention introduced a new concept both in clinical practice and experimental approaches. While single agent ER-independent chemopreventive therapy based on RXR-selective retinoids and other agents has produced promising results, even minor toxicity remains an obstacle to their clinical use. We performed unbiased high throughput screening to identify synergistic growth suppressive combinations of agents in normal mammary epithelial cells. A RXR-selective agonist and a beta-adrenergic inhibitor, as a candidate combination, at low concentrations demonstrated marked cancer preventive activity in a mouse model of Her2-induced ER-negative breast cancer. To elucidate the molecular mechanisms of this chemopreventive activity proteomic analyses of the candidate drug combination ensued. These revealed increased levels and phosphorylation of the DNA damage sensor ATM, along with elevated levels of the ARID1A subunit of the SWI/SNF chromatin remodeler in normal breast epithelial cells, but not in in situ breast carcinoma cells. Thus, we hypothesized that the maintenance of genomic integrity through ARID1A is a critical factor in the effectiveness of cancer preventive agents and helps reduce the odds of transformation. Genomic regions of increased accessibility are mapped by high throughput sequencing enabling multidimensional assays of the regulatory chromatin regions. Enhancers identified through chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) for H3K27 acetyl complemented with histones H3K4 will be discussed. We propose that ARID1A may mediate antiproliferative effects through genome-wide pausing of transcription of cell cycle regulatory genes. These results will help establish a role for the crosstalk of rexinoid and adrenergic signaling in the maintenance of a conserved chromatin remodeling mechanism and thus, premalignancy in breast epithelial cells. Citation Format: Sham Jdeed, Balint L. Balint, Iván P. Uray. Genomic regulation of the cell cycle as a function of ARID1A occupancy in breast epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5068.

Abstract 971: PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells

Abstract Protein arginine methyltransferase 5 (PRMT5) has recently been identified as a promising therapeutic target in various solid and hematologic cancers, while PRMT5 inhibitors are currently in clinical development. Functionally, PRMT5 has an established role in regulating splicing, and is essential for hematopoiesis. However, the mechanisms underlying PRMT5 dependency in HSCs, particularly PRMT5-mediated splicing, remain unclear. Moreover, how pharmacological inhibition of PRMT5 affects HSCs remains undetermined. To clarify the function of PRMT5 in HSCs, we adopted a loss-of-function approach, coupled with functional and transcriptome profiling. We report that depletion of PRMT5 activity severely perturbs HSC maintenance. HSCs from Mx1-Cre Prmt5fl/fl mice (PRMT5Δ/Δ), which exhibit severe bone marrow (BM) failure, failed to reconstitute lethally irradiated wild-type recipients. Notably, this was associated with rapid loss of Prmt5Δ/Δ HSCs. Similarly, EPZ015666-mediated PRMT5 inhibition led to a dose-dependent decrease in the growth and viability of EML cells (murine BM-derived hematopoietic precursor cell line). Interestingly, this was associated with increased HSC size and PI3K/AKT/mTOR pathway activity. In addition, protein synthesis rate was also elevated in an mTOR-dependent manner. Thus, we confirm the cell-intrinsic requirement for PRMT5 activity in HSCs, and demonstrate a potential role for PRMT5 in regulating HSC proteostasis. We also analyzed the splice variant landscape of PRMT5Δ/Δ HSCs, and observed substantial upregulation of intron retention and exon skipping events; which were overrepresented for processes associated with maintenance of genomic integrity. Notably, we show that splicing of key genes involved in base excision repair, telomere maintenance and the Fanconi anemia pathway were dysregulated in PRMT5Δ/Δ HSCs, as well as EPZ015666-treated EML cells. Thus, suggesting that HSCs depleted of PRMT5 activity are potentially deficient in DNA repair. In agreement, PRMT5Δ/Δ HSCs exhibited elevated γH2A.X levels, replicative stress-associated oxidative DNA lesions and DNA strand breaks. Likewise, EPZ015666-treated EML cells exhibited dose-dependent increments in γH2A.X levels, and increased sensitivity to DNA damaging agents. Corroborating these observations, loss of PRMT5 activity in both PRMT5Δ/Δ HSCs and EPZ015666-treated EML cells led to elevated reactive oxygen species (ROS) levels, p53 activation and induction of apoptosis; which was significantly rescued by antioxidant treatment or co-depletion of p53. Therefore, depletion of PRMT5 activity renders HSCs vulnerable to DNA damage. Collectively, our findings uncover a critical role for PRMT5 in maintaining genomic integrity, and highlight a novel link between PRMT5 and proteostasis in HSCs. Citation Format: Darren Qiancheng Tan, Ying Li, Chong Yang, Jia Li, Shi Hao Tan, Desmond W. Chin, Ayako Nakamura-Ishizu, Henry Yang, Toshio Suda. PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 971.

Abstract 2574: Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability

Abstract Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors. Citation Format: Lorn Kategaya, Senthil K. Perumal, Jeffrey H. Hager, Lisa D. Belmont. Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2574.

Abstract 1740: Characterization of CDK9 role in the DNA damage response

Abstract Introduction: The DNA damage response (DDR) is a well-coordinated pathway capable of sensing and repairing different types of DNA damage. Double-strand break (DSB) lesions activate the ATM-CHK2 axis, which is responsible for the cell cycle arrest, prompting cells for DNA repair. DSBs are primarily repaired through two distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1 play an important role in DDR by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified the putative interaction of the cyclin-dependent kinase 9 (CDK9) with BRCA1 and BARD1 (BRCA1-associated RING domain 1). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. More recently, we characterized CDK9 as a DDR player by modulating BRCA1 recruitment to DNA damaged sites and consequently the HR pathway. We also showed that cells lacking CDK9 failure to form RAD51, but not 53BP1, ionizing radiation-induced foci (IRIF) and are characterized by an increased sensitivity to DSBs Methods and Results: Here we show that the CDK9-BRCA1 interaction occurs through BRCA1 N-terminal region in a BRCA1-BARD1 heterodimer formation-dependent manner. We also identified a CDK9 mutant (E369A) that abrogates the CDK9-BRCA1 interaction (through BRCA1 C-terminal region). Moreover, we characterized CHK2 as a novel CDK9-interaction partner, through ectopic GST-pulldown and constitutive co-immunoprecipitation assays. Further, cells lacking CDK9 are deficient in promoting G2/M arrest after IR-induced DNA damage. We also identified new CDK9 interactions in a DDR-related context using a TAP-MS approach. Conclusion: Collectively, our data suggest a role for CDK9 in the DDR through DNA damage repair and cell cycle checkpoint regulation Citation Format: Thales C. Nepomuceno, Guilherme Suarez-Kurtz, Alvaro N. Monteiro, Marcelo A. Carvalho. Characterization of CDK9 role in the DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1740.

Holocaust history is not reflected in telomere homeostasis in survivors and their offspring

Telomeres, nucleoprotein structures at the ends of eukaryotic chromosomes, are crucial for the maintenance of genome integrity. While the lengths of telomeres at birth are determined genetically, many factors including environmental and living conditions affect the telomere lengths during a lifespan. In this context, extreme and long-term stress has been shown to negatively impact telomeres and their protective function, with even offspring being influenced by the stress experienced by parents. Using quantitative PCR, the relative lengths of telomeres of survivors of the Holocaust during World War II and two generations of their offspring were analyzed. These data were related to those of control groups, persons of comparable age without a strong life stress experience. In contrast to previous studies of other stress-exposed groups, the relative lengths of telomeres were comparable in groups of persons exposed to Holocaust-related stress and their progenies, and in control groups. Interestingly, shorter telomeres of Holocaust survivors of the age under 12 in the year 1945 compared to Holocaust survivors of the age above 12 were detected. Our results are discussed with respect to certain exceptionality of persons having been able to cope with an extreme stress more than 70 years ago and living to a very old age.

SIRT1 modulates cell cycle progression by regulating CHK2 acetylation\u2212phosphorylation

Both the stress-response protein, SIRT1, and the cell cycle checkpoint kinase, CHK2, play critical roles in aging and cancer via the modulation of cellular homeostasis and the maintenance of genomic integrity. However, the underlying mechanism linking the two pathways remains elusive. Here, we show that SIRT1 functions as a modifier of CHK2 in cell cycle control. Specifically, SIRT1 interacts with CHK2 and deacetylates it at lysine 520 residue, which suppresses CHK2 phosphorylation, dimerization, and thus activation. SIRT1 depletion induces CHK2 hyperactivation-mediated cell cycle arrest and subsequent cell death. In vivo, genetic deletion of Chk2 rescues the neonatal lethality of Sirt1−/− mice, consistent with the role of SIRT1 in preventing CHK2 hyperactivation. Together, these results suggest that CHK2 mediates the function of SIRT1 in cell cycle progression, and may provide new insights into modulating cellular homeostasis and maintaining genomic integrity in the prevention of aging and cancer.

Genomic instability and innate immune responses to self-DNA in progeria.

In the last decade, we have seen increasing evidence of the importance of structural nuclear proteins such as lamins in nuclear architecture and compartmentalization of genome function and in the maintenance of mechanical stability and genome integrity. With over 400 mutations identified in the LMNA gene (encoding for A-type lamins) associated with more than ten distinct degenerative disorders, the role of lamins as genome caretakers and the contribution of lamins dysfunction to disease are unarguable. However, the molecular mechanisms whereby lamins mutations cause pathologies remain less understood. Here, we review pathways and mechanisms recently identified as playing a role in the pathophysiology of laminopathies, with special emphasis in Hutchinson Gilford Progeria Syndrome (HGPS). This devastating incurable accelerated aging disease is caused by a silent mutation in the LMNA gene that generates a truncated lamin A protein "progerin" that exerts profound cellular toxicity and organismal decline. Patients usually die in their teens due to cardiovascular complications such as myocardial infarction or stroke. To date, there are no efficient therapies that ameliorate disease progression, stressing the need to understand molecularly disease mechanisms that can be targeted therapeutically. We will summarize data supporting that replication stress is a major cause of genomic instability in laminopathies, which contributes to the activation of innate immune responses to self-DNA that in turn accelerate the aging process.

The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain.

Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

Effects of environmental pollution on the rDNAomics of Amazonian fish

Pollution is a growing environmental problem throughout the world, and the impact of human activities on biodiversity and the genetic variability of natural populations is increasingly preoccupying, given that adaptive processes depend on this variability, in particular that found in the repetitive DNA. In the present study, the mitochondrial DNA (COI) and the distribution of repetitive DNA sequences (18S and 5S rDNA) in the fish genome were analysed in fish populations inhabiting both polluted and unpolluted waters in the northern Amazon basin. The results indicate highly complex ribosomal sequences in the fish genome from the polluted environment because these sequences are involved primarily in the maintenance of genome integrity, mediated by a systematic increase in the number of copies of the ribosomal DNA in response to changes in environmental conditions.

Early neuronal accumulation of DNA double strand breaks in Alzheimer\u2019s disease

The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer’s disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX—a post-translational histone modification that is widely used as a marker of DSBs—revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease.

The Plant DNA Damage Response: Signaling Pathways Leading to Growth Inhibition and Putative Role in Response to Stress Conditions.

Maintenance of genome integrity is a key issue for all living organisms. Cells are constantly exposed to DNA damage due to replication or transcription, cellular metabolic activities leading to the production of Reactive Oxygen Species (ROS) or even exposure to DNA damaging agents such as UV light. However, genomes remain extremely stable, thanks to the permanent repair of DNA lesions. One key mechanism contributing to genome stability is the DNA Damage Response (DDR) that activates DNA repair pathways, and in the case of proliferating cells, stops cell division until DNA repair is complete. The signaling mechanisms of the DDR are quite well conserved between organisms including in plants where they have been investigated into detail over the past 20 years. In this review we summarize the acquired knowledge and recent advances regarding the DDR control of cell cycle progression. Studying the plant DDR is particularly interesting because of their mode of development and lifestyle. Indeed, plants develop largely post-embryonically, and form new organs through the activity of meristems in which cells retain the ability to proliferate. In addition, they are sessile organisms that are permanently exposed to adverse conditions that could potentially induce DNA damage in all cell types including meristems. In the second part of the review we discuss the recent findings connecting the plant DDR to responses to biotic and abiotic stresses.

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens.

The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells.

Astral microtubule forces alter nuclear organization and inhibit DNA repair in budding yeast.

Dividing cells must balance the maintenance of genome integrity with the generation of cytoskeletal forces that control chromosome position. In this study, we investigate how forces on astral microtubules impact the genome during cell division by using live-cell imaging of the cytoskeleton, chromatin, and DNA damage repair in budding yeast. Our results demonstrate that dynein-dependent forces on astral microtubules are propagated through the spindle during nuclear migration and when in excess can increase the frequency of double-stranded breaks (DSBs). Under these conditions, we find that homology-directed repair of DSBs is delayed, indicating antagonism between nuclear migration and the mechanism of homology-directed repair. These effects are partially rescued by mutants that weaken pericentric cohesion or mutants that decrease constriction on the nucleus as it moves through the bud neck. We propose that minimizing nuclear movement aids in finding a donor strand for homologous recombination.

Integrative rDNAomics-Importance of the Oldest Repetitive Fraction of the Eukaryote Genome.

Nuclear ribosomal RNA (rRNA) genes represent the oldest repetitive fraction universal to all eukaryotic genomes. Their deeply anchored universality and omnipresence during eukaryotic evolution reflects in multiple roles and functions reaching far beyond ribosomal synthesis. Merely the copy number of non-transcribed rRNA genes is involved in mechanisms governing e.g., maintenance of genome integrity and control of cellular aging. Their copy number can vary in response to environmental cues, in cellular stress sensing, in development of cancer and other diseases. While reaching hundreds of copies in humans, there are records of up to 20,000 copies in fish and frogs and even 400,000 copies in ciliates forming thus a literal subgenome or an rDNAome within the genome. From the compositional and evolutionary dynamics viewpoint, the precursor 45S rDNA represents universally GC-enriched, highly recombining and homogenized regions. Hence, it is not accidental that both rDNA sequence and the corresponding rRNA secondary structure belong to established phylogenetic markers broadly used to infer phylogeny on multiple taxonomical levels including species delimitation. However, these multiple roles of rDNAs have been treated and discussed as being separate and independent from each other. Here, I aim to address nuclear rDNAs in an integrative approach to better assess the complexity of rDNA importance in the evolutionary context.

The SMC5/6 Complex Subunit NSE4A Is Involved in DNA Damage Repair and Seed Development.

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

Centrosomal Actin Assembly Is Required for Proper Mitotic Spindle Formation and Chromosome Congression

SummaryCytoskeletal cross talk between actin filaments and microtubules is a common mechanism governing the assembly of cellular structures, i.e., during filopodia formation or cilia organization. However, potential actin-microtubule interactions during mammalian cell divisions are less well understood. At mitotic entry, centrosomes propagate the formation of the mitotic spindle, thereby aligning individual chromosomes to the metaphase plate, a process coined chromosome congression. Here, we identify actin filament assembly spatially defined at centrosomes contemporaneously with spindle microtubules forming during prometaphase. We show that pharmacological Arp2/3 complex inhibition as well as overexpression of the Arp2/3 complex inhibitory protein Arpin decreased spindle actin. As a consequence, mitotic spindle formation is impaired, which resulted in disorganized chromosome congression and ultimately mitotic defects in non-transformed cells. Thus centrosomal Arp2/3 complex activity plays a role in the maintenance of genomic integrity during mitosis.

Inhibition of Wee1 sensitizes AML cells to ATR inhibitor VE-822-induced DNA damage and apoptosis

Resistance to standard induction therapy and relapse remain the primary challenges for improving therapeutic effects in acute myeloid leukemia (AML); thus, novel therapeutic strategies are urgently required. Ataxia telangiectasia and Rad3-related protein (ATR) is a key regulator of different types of DNA damage, which is crucial for the maintenance of genomic integrity. The ATR-selective inhibitor VE-822 has proper solubility, potency, and pharmacokinetic properties. In this study, we investigated the anti-leukemic effects of VE-822 alone or combined with Wee1-selective inhibitor AZD1775 in AML cells. Our results showed that VE-822 inhibited AML cell proliferation and induced apoptosis in a dose-dependent manner. AZD1775 significantly promoted VE-822-induced inhibition of AML cell proliferation and led to a decreased number of cells in the G2/M phase. VE-822 and AZD1775 decreased the protein levels of ribonucleotide reductase M1 (RRM1) and M2 (RRM2) subunits, key enzymes in the synthesis of deoxyribonucleoside triphosphate, which increased DNA replication stress. VE-822 combined with AZD1775 synergistically induced AML cell apoptosis and led to replication stress and DNA damage in AML cell lines. Our study demonstrated that AZD1775 synergistically promotes VE-822-induced anti-leukemic activity in AML cell lines and provides support for clinical research on VE-822 in combination with AZD1775 for the treatment of AML patients.

Nuclear actin interactome analysis links actin to KAT14 histone acetyl transferase and mRNA splicing.

ABSTRACTIn addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here, we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID, to identify binding partners for nuclear actin. Common high-confidence interactions highlight the role of actin in chromatin-remodeling complexes and identify the histone-modifying complex human Ada-Two-A-containing (hATAC) as a novel actin-containing nuclear complex. Actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. Transient interactions detected through BioID link actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative splicing in minigene assays, likely by affecting the transcription elongation rate. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes.This article has an associated First Person interview with the first author of the paper.