scholarly journals Deletions of 5q Promote Genome Instability in TP53-Mutant MDS/AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1244-1244
Author(s):  
Andreea Reilly ◽  
Stephanie Busch ◽  
Janis L. Abkowitz ◽  
Pamela S. Becker ◽  
Sergei Doulatov
Keyword(s):  
Dna Damage ◽  
Chromosomal Instability ◽  
Molecular Mechanisms ◽  
Genome Instability ◽  
Dna Damage Repair ◽  
Normal Karyotype ◽  
Complex Karyotype ◽  
Differentiation Potential ◽  
Tp53 Mutations ◽  
Damage Repair

Purpose TP53 mutations in myeloid neoplasms (MDS/AML) are associated with high-risk disease, poor outcome, and complex karyotype. The molecular mechanisms which lead to global chromosomal instability remain poorly understood. Loss of 5q [del(5q)] is the most frequent cytogenetic abnormality associated with TP53 mutations suggesting that haploinsufficiency of genes on 5q contributes to chromosomal instability. Methods We reprogrammed MDS/AML patient samples to establish genetically accurate iPSC lines from preleukemic subclones. We generated iPSCs with TP53 mutations and del(5q), differentiated them to hematopoietic progenitors (HPCs), and determined the contribution of del(5q) to genome instability. Results By reprogramming MDS/AML complex karyotype patient samples, we identified iPSCs with heterozygous TP53 mutations (TP53-only), as well as iPSCs with TP53 mutations and del5(q22-q31) (TP53;del5q), and an otherwise normal karyotype. HPCs derived from TP53;del5q iPSCs had decreased multilineage differentiation potential compared to the TP53-only HPCs. Gene expression analysis of TP53;del5q HPCs revealed downregulation of genes involved in chromosome segregation and DNA damage repair. Following irradiation TP53;del5q cells had significantly delayed DNA damage repair kinetics. In order to evaluate the effects of TP53 and del(5q) on chromosomal segregation during stress, we arrested the cells in mitosis by disrupting the mitotic spindle and quantified the induction of micronuclei, a marker of chromosomal instability that occurs due to lagging chromosomes. TP53;del5q cells had an increased frequency of micronuclei formation compared to TP53-only cells. We also detected micronuclei in primary AML patient samples. Micronuclei in iPSC-HPCs and primary patient cells had disrupted nuclear envelope and DNA damage marked by y-H2AX. Conclusions Our reprogramming approach revealed that TP53 mutations are disease-initiating and frequently followed by 5q loss. We propose that del(5q) cooperates with mutant TP53 to promote genome instability via two distinct mechanisms: classical double-stranded break repair and micronuclei formation. The latter is associated with global chromosomal instability, aneuploidy, and chromothripsis. We propose that loss of 5q accelerates genome instability in TP53-mutant cells which over time impedes normal hematopoietic differentiation and leads to complex karyotype. Disclosures Becker: The France Foundation: Honoraria; Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding.

scholarly journals Tumor Cell–Accelerated Senescence Is Associated With DNA-PKcs Status and Telomere Dysfunction Induced by Radiation

Dose-Response ◽  
2018 ◽  
Vol 16 (2) ◽  
pp. 155932581877152 ◽  
Author(s):  
Miaomiao Zhang ◽  
Xiaopeng Guo ◽  
Yue Gao ◽  
Dong Lu ◽  
Wenjian Li
Keyword(s):  
Dna Damage ◽  
Real Time ◽  
Genome Instability ◽  
Dna Damage Repair ◽  
Cancer Cell Line ◽  
Histochemical Staining ◽  
Damage Repair ◽  
Radiation Induced ◽  
Accelerated Senescence ◽  
Mcf 7

Whether telomere structure integrity is related to radiosensitivity is not well investigated thus far. In this study, we investigated the relation between telomere instability and radiation-induced accelerated senescence. Partial knockdown of DNA-dependent catalytic subunit of protein kinase (DNA-PKcs) in human breast cancer cell line MCF-7 was established by small interfering RNA. Radiosensitivity of control and DNA-PKcs knockdown MCF-7 cells was analyzed by clonogenetic assay. Cell growth was measured by real-time cell electronic sensing. Senescence and apoptosis were evaluated by β-galactosidase histochemical staining and fluorescence-activated cell sorting, respectively. DNA damage was determined by long polymerase chain reaction (PCR). Telomere length and integrity were analyzed by real-time PCR and cytogenetic assay, respectively. DNA-PKcs knockdown MCF-7 cells were more sensitive to X-irradiation than control cells. Further investigation revealed that accelerated senescence is more pronounced than apoptosis in cells after radiation, particularly in DNA-PKcs knockdown cells. The cytogenetic assay and kinetics of DNA damage repair revealed that the role of telomere end-capping in DNA-PKcs, rather than DNA damage repair, was more relevant to radiosensitivity. To our knowledge, this is the first study to show that DNA-PKcs plays an important role in radiation-induced accelerated senescence via maintenance of telomere integrity in MCF-7 cells. These results could be useful for future understanding of the radiation-induced genome instability and its consequences.

Genomic characterization of brain metastases (BM) in high-grade serous ovarian cancer (HGSOC).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13580-e13580
Author(s):  
Renata Duchnowska ◽  
Anna Maria Supernat ◽  
Rafał Pęksa ◽  
Marta Łukasiewicz ◽  
Tomasz Stokowy ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Dna Damage ◽  
Somatic Mutations ◽  
Dna Damage Repair ◽  
Genetic Alterations ◽  
High Grade ◽  
Serous Ovarian Cancer ◽  
Primary Tumors ◽  
Tp53 Mutations ◽  
Damage Repair

e13580 Background: BM are a rare occurrence in ovarian cancer (OC) and their molecular characteristics is virtually unknown. DNA damage repair (DDR) deficiency is prevalent in OC, and co-mutated TP53 and any DDR denotes high tumor mutation burden (TMB). We genetically characterized a unique series of high-grade serous ovarian cancer (HGSOC) patients who developed BM to identify alterations of potential clinical relevance. Methods: Whole-exome sequencing (2x150bp, SureSelectXT Library Prep Kit, Illumina’s NovaSeq platform) was performed in matched BM, primary tumors (PT) and normal tissue. DNA was extracted from FFPE samples using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany). All mutations were checked with Catalogue of Somatic Mutations in Cancer (COSMIC) and Integrative Genomics Viewer (IGV). Results: Study group included 10 HGSOC patients (International Federation of Gynecology and Obstetrics classification (FIGO) II-IV, mean age at diagnosis 48 years, range 35-59). Median time from primary HGSOC diagnosis to BM was 38 months (range, 18 to 149). TP53 somatic mutations were found in both primary tumor (PT) and BM in 8 patients. The other 2 cases harbored TP53 mutations not reported in COSMIC catalogue: p.S60L and intronic TP53 mutation preceding p.I322 (IGV). In 9 cases TP53 mutations coexisted with germline or somatic DNA damage repair deficiency. Four cases contained BRCA1 mutations (all germline), and none harbored germline BRCA2 mutation. Other mutated genes included MLH1 (2 somatic, 2 germline), ATR (4 germline, 1 somatic), AMT (1 somatic), RAD50 (1 somatic), ERCC4 (1 somatic), FANCD2 (1 somatic) and RPA1 (1 germline). Three mutation signatures defined in the COSMIC database were indentified in BM: 6, 20 and 30. In 6 cases these mutations were shared in PT, and in another 4 their presence in PT could not be determined due to technical reasons. Median survival from BM was 31 months (range, 5 to 184). Conclusions: Genomic analysis of BM provides an opportunity to identify potentially clinically informative alterations. Mutational profiles in PT are generally reflected in BM. Detected genetic alterations suggest their potential sensitivity to PARP inhibitors and immunotherapy.

scholarly journals Recruitment of the Type B Histone Acetyltransferase Hat1p to Chromatin Is Linked to DNA Double-Strand Breaks

2006 ◽  
Vol 26 (9) ◽  
pp. 3649-3658 ◽  
Author(s):  
Song Qin ◽  
Mark R. Parthun
Keyword(s):  
Dna Damage ◽  
Molecular Mechanisms ◽  
Dna Damage Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Similar Distribution ◽  
Tail Domain ◽  
Type B ◽  
Direct Role ◽  
Damage Repair

ABSTRACT Type B histone acetyltransferases are thought to catalyze the acetylation of the NH2-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH2-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.

Contribution of DNA damage repair gene mutations and concomitant TP53 variations to the efficacy of platinum-based chemotherapy as initial therapeutic option in Chinese NSCLC patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e21002-e21002
Author(s):  
Ke Ma ◽  
Jun Peng ◽  
Huachuan Zhang ◽  
Jintao He ◽  
Bo Xiao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Disease Progression ◽  
Cox Regression ◽  
Dna Damage Repair ◽  
Cox Regression Analysis ◽  
Tp53 Mutations ◽  
Damage Repair ◽  
Platinum Based Chemotherapy ◽  
Risk Of Disease ◽  
Nsclc Patients

e21002 Background: Though targeted, immune-, even combinational therapies have been applied widely, platinum-based chemotherapy still plays irreplaceable role as initial management for patient with either resected or advanced NSCLC without actionable target. Platinum is sought to increase DNA damage burden inducing cell death, especially in cells with intrinsic DNA damage repair (DDR) dysfunction. This study aims to explore impact of DDR genes as well as co-occurrence of TP53 mutations on prognosis of NSCLC patients. Methods: 110 NSCLC patients were consecutively recruited from 2016-2018. 508 cancer related genes were sequenced by BGI-seq 500. COX regression was used to evaluate risk of disease progression as well as cancer-related death among patients with various DDR/TP53 status. Results: 61 NSCLC patients with stage II-IV (median age of onset: 55 years) were recruited who received platinum-based therapy as adjuvant or 1st line systemic therapy. Till 15th.Oct.2019, median follow-up time was 24 months. 21 patients (34.4% of 61) carried DDR mutations. TP53 mutations were detected in 36 patients (59% of 61). 11 patients (18% of 61) displayed TP53/DDR co-mutations. COX regression analysis indicated NSCLC patients with DDR mutation would have lower risk of disease progression (HR:0.37, P = 0.19) as well as superior overall survival time (HR:0.35, P = 0.035). Patients with DDR co-mutations had no superior survival benefit. Patients with HRR mutations presented similar result. Patients with TP53 mutations had similar risk as ones with other mutations. While those with DDR concomitant mutations showed significantly improved clinical benefit (HR for PFS:0.28, P = 0.05; HR for OS:0.22, P = 0.023). Conclusions: DNA damage repair genes in Chinese NSCLC patients may play vital roles in predicting efficacy of platinum-based chemotherapy even in the context of TP53 co-mutation. Further, combination of BRCA2 and PALB2 may be better associated with patients’ prognosis. Large cohort should be set up to validate this finding in near future. [Table: see text]

scholarly journals Interactome Analysis Reveals a Novel Role for RAD6 in the Regulation of Proteasome Activity and Localization in Response to DNA Damage

2016 ◽  
Vol 37 (6) ◽  
Author(s):  
Hongli An ◽  
Lu Yang ◽  
Chen Wang ◽  
Zhixue Gan ◽  
Haihui Gu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Dna Damage Repair ◽  
Repair Process ◽  
Proteasome Activity ◽  
Nuclear Antigen ◽  
Ppi Networks ◽  
Damage Repair ◽  
Repair Pathways

ABSTRACT RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.

scholarly journals DNA Damage Repair Interference By WEE1 Inhibition with AZD1775 Overcomes Combined Azacitidine and Venetoclax Resistance in Acute Myeloid Leukmeia (AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2559-2559
Author(s):  
Raoul Tibes ◽  
Diego Ferreira Coutinho ◽  
Michael Tuen Tuen ◽  
Xufeng Chen ◽  
Christina Glytsou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Low Dose ◽  
Dna Damage Repair ◽  
Single Agent ◽  
Leukemic Cells ◽  
Damage Repair ◽  
Arac Treatment ◽  
Acute Myeloid

Acute myeloid leukemia (AML) has remained one of the most treatment resistant and deadliest cancers. The survival of AML blast cells is controlled by the balance of anti- and pro-apoptotic proteins. Recently approved Bcl-2 targeted therapy of AML with the Bcl-2 specific inhibitor Venetoclax in combinations has improved patients outcomes. However, a priori and developing resistance to venetoclax combinations with hypomethylating agents (HMA) azacitidine and decitabine challenge this treatment. As such, novel therapies to overcome venetoclax-HMA resistance are urgently needed. We have identified a combination of DNA damage repair interference by WEE1 inhibition with AZD1775, combined with low dose cytarabine (AraC) as an effective strategy to overcome combined venetoclax-azacitidine resistance (VAR). AZD1775 with low dose AraC induced massive apoptosis (by Annexin V and cleaved caspase-3) and almost completely reduced viability and clonogenic growth of primary AML cells. To delineate the molecular mechanism of the synergistic effect of AZD1775/AraC we performed RNAseq analysis of single agent or the combination of AZD1775+AraC in AML cell lines and primary CD34+ selected AML patient cells with the goal to identify deferentially regulated genes indicating a mechanistic underpinning of the potent activity. Only 2 genes were deferentially regulated across cell lines and CD34+ selected cells under AZD1775+AraC treatment: one of these is NR4A1, an orphan nuclear receptor, which we went on to validate as a potential downstream target of Wee1 inhibition. The inactivation of NR4A1 in mice was previously shown to induce AML and to maintain leukemia stem cells. Using qPCR we confirmed that the expression of NR4A1 is upregulated after AZD1775/AraC combo treatment in human leukemic cells. We then demonstrated that activators of NR4A1 (cytosporone B and pPhOCH3) reduce viability of leukemic cells, while NR4A1 inhibitor pPhOH was able to abolish the effect of AZD1775/AraC combo treatment increasing leukemic cell viability]. To investigate the involvement of mitochondria in the effect of AZD1775/AraC treatment we performed the expression of mitochondrial genes and pathway analyses in RNAseq data and found that mitochondrial gene expression, including many genes involved in apoptosis, has most dramatic changes in the combo treatment if compared to the single agents. Subsequently, we have examined the expression of the main BCL-2 family apoptotic genes by qPCR and western blot analysis. We found that AZD1775/AraC induces the expression of Bim isoforms, whereas Bcl-2, Mcl-1 and Bcl-Xl were largely unaffected. NR4A1 was previously shown to translocate to mitochondria, release Bim from Bcl-2 protein binding, as well as convert Bcl-2 to an extreme potent pro-apoptotic form. Finally, we generated several additional VAR cell lines and cells with subclones and demonstrated that AZD1775/AraC combination treatment is able to overcome VAR in almost every clone. Our results show that DNA damage repair interference with Wee1 inhibition has the potential to overcome VAR through a novel mechanisms of AZD1775 increasing NR4A1, freeing pro-apoptotic Bim irrespective of anti-apoptotic Bcl-2 proteins leading to massive apoptotic cell death in AML cells. The precise molecular mechanisms and the involvement of NR4A1 in this phenomenon will be presented at the meeting. Our findings will help to develop new therapeutic strategies in AML treatment and a trial of AZD1775 + AraC in AML is currently ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals DNA Damage Repair Profiles Alteration Characterize a Hepatocellular Carcinoma Subtype With Unique Molecular and Clinicopathologic Features

2021 ◽  
Vol 12 ◽  
Author(s):  
Peng Lin ◽  
Rui-zhi Gao ◽  
Rong Wen ◽  
Yun He ◽  
Hong Yang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Damage ◽  
Molecular Mechanisms ◽  
Dna Damage Repair ◽  
Clinicopathological Features ◽  
Molecular Characteristics ◽  
Molecular Features ◽  
Damage Repair ◽  
Molecular Phenotypes ◽  
Aggressive Clinical Behavior

Hepatocellular carcinoma (HCC) is one of the most common malignancies and displays high heterogeneity of molecular phenotypes. We investigated DNA damage repair (DDR) alterations in HCC by integrating multi-omics data. HCC patients were classified into two heterogeneous subtypes with distinct clinical and molecular features: the DDR-activated subtype and the DDR-suppressed subtype. The DDR-activated subgroup is characterized by inferior prognosis and clinicopathological features that result in aggressive clinical behavior. Tumors of the DDR-suppressed class, which have distinct clinical and molecular characteristics, tend to have superior survival. A DDR subtype signature was ultimately generated to enable HCC DDR classification, and the results were confirmed by using multi-layer date cohorts. Furthermore, immune profiles and immunotherapy responses are also different between the two DDR subtypes. Altogether, this study illustrates the DDR heterogeneity of HCCs and is helpful to the understanding of personalized clinicopathological and molecular mechanisms responsible for unique tumor DDR profiles.

Ubiquitin-like proteins in the DNA damage response: the next generation

2020 ◽  
Vol 64 (5) ◽  
pp. 737-752 ◽  
Author(s):  
Isabelle C. Da Costa ◽  
Christine K. Schmidt
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Molecular Mechanisms ◽  
Genome Instability ◽  
Cellular Responses ◽  
Human Diseases ◽  
Current Status ◽  
Post Translational Modifications ◽  
Damage Repair ◽  
Damage Response

Abstract DNA suffers constant insult from a variety of endogenous and exogenous sources. To deal with the arising lesions, cells have evolved complex and coordinated pathways, collectively termed the DNA damage response (DDR). Importantly, an improper DDR can lead to genome instability, premature ageing and human diseases, including cancer as well as neurodegenerative disorders. As a crucial process for cell survival, regulation of the DDR is multi-layered and includes several post-translational modifications. Since the discovery of ubiquitin in 1975 and the ubiquitylation cascade in the early 1980s, a number of ubiquitin-like proteins (UBLs) have been identified as post-translational modifiers. However, while the importance of ubiquitin and the UBLs SUMO and NEDD8 in DNA damage repair and signalling is well established, the roles of the remaining UBLs in the DDR are only starting to be uncovered. Herein, we revise the current status of the UBLs ISG15, UBL5, FAT10 and UFM1 as emerging co-regulators of DDR processes. In fact, it is becoming clear that these post-translational modifiers play important pleiotropic roles in DNA damage and/or associated stress-related cellular responses. Expanding our understanding of the molecular mechanisms underlying these emerging UBL functions will be fundamental for enhancing our knowledge of the DDR and potentially provide new therapeutic strategies for various human diseases including cancer.

scholarly journals A Link between Replicative Stress, Lamin Proteins, and Inflammation

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 552
Author(s):  
Simon Willaume ◽  
Emilie Rass ◽  
Paula Fontanilla-Ramirez ◽  
Angela Moussa ◽  
Paul Wanschoor ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Molecular Mechanisms ◽  
Genome Instability ◽  
Inflammatory Diseases ◽  
Dna Lesions ◽  
Inflammatory Factors ◽  
Replicative Stress ◽  
Damage Repair ◽  
Beneficial Response

Double-stranded breaks (DSB), the most toxic DNA lesions, are either a consequence of cellular metabolism, programmed as in during V(D)J recombination, or induced by anti-tumoral therapies or accidental genotoxic exposure. One origin of DSB sources is replicative stress, a major source of genome instability, especially when the integrity of the replication forks is not properly guaranteed. To complete stalled replication, restarting the fork requires complex molecular mechanisms, such as protection, remodeling, and processing. Recently, a link has been made between DNA damage accumulation and inflammation. Indeed, defects in DNA repair or in replication can lead to the release of DNA fragments in the cytosol. The recognition of this self-DNA by DNA sensors leads to the production of inflammatory factors. This beneficial response activating an innate immune response and destruction of cells bearing DNA damage may be considered as a novel part of DNA damage response. However, upon accumulation of DNA damage, a chronic inflammatory cellular microenvironment may lead to inflammatory pathologies, aging, and progression of tumor cells. Progress in understanding the molecular mechanisms of DNA damage repair, replication stress, and cytosolic DNA production would allow to propose new therapeutical strategies against cancer or inflammatory diseases associated with aging. In this review, we describe the mechanisms involved in DSB repair, the replicative stress management, and its consequences. We also focus on new emerging links between key components of the nuclear envelope, the lamins, and DNA repair, management of replicative stress, and inflammation.

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