15 results on '"Barreto-Galvez A"'
Search Results
2. Functional mapping of PHF6 complexes in chromatin remodeling, replication dynamics, and DNA repair
- Author
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Alvarez, Silvia, da Silva Almeida, Ana C., Albero, Robert, Biswas, Mayukh, Barreto-Galvez, Angelica, Gunning, Thomas S., Shaikh, Anam, Aparicio, Tomas, Wendorff, Agnieszka, Piovan, Erich, Van Vlierberghe, Pieter, Gygi, Steven, Gautier, Jean, Madireddy, Advaitha, and Ferrando, Adolfo A.
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- 2022
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3. High burden of clonal hematopoiesis in first responders exposed to the World Trade Center disaster
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Jasra, Sakshi, Giricz, Orsi, Zeig-Owens, Rachel, Pradhan, Kith, Goldfarb, David G., Barreto-Galvez, Angelica, Silver, Alexander J., Chen, Jiahao, Sahu, Srabani, Gordon-Mitchell, Shanisha, Choudhary, Gaurav S., Aluri, Srinivas, Bhagat, Tushar D., Shastri, Aditi, Bejan, Cosmin A., Stockton, Shannon S., Spaulding, Travis P., Thiruthuvanathan, Victor, Goto, Hiroki, Gerhardt, Jeannine, Haider, Syed Hissam, Veerappan, Arul, Bartenstein, Matthias, Nwankwo, George, Landgren, Ola, Weiden, Michael D., Lekostaj, Jacqueline, Bender, Ryan, Fletcher, Frederick, Greenberger, Lee, Ebert, Benjamin L., Steidl, Ulrich, Will, Britta, Nolan, Anna, Madireddy, Advaitha, Savona, Michael R., Prezant, David J., and Verma, Amit
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- 2022
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4. Acetyl transferase EP300 deficiency leads to chronic replication stress mediated by defective fork protection at stalled replication forks
- Author
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Barreto-Galvez, Angelica, primary, Niljikar, Mrunmai, additional, Gagliardi, Julia, additional, Zhang, Ranran, additional, Juwarwala, Aastha, additional, Pradeep, Archana, additional, Kumar, Vasudha, additional, Tiwari, Priyanka, additional, Shaikh, Anam, additional, Sharma, Kritika, additional, Gerhardt, Jeannine, additional, Cao, Jian, additional, Kataoka, Keisuke, additional, Durbin, Adam, additional, Qi, Jun, additional, Ye, B Hilda, additional, and Madireddy, Advaitha, additional
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- 2023
- Full Text
- View/download PDF
5. Acetyl transferase EP300 deficiency leads to chronic replication stress mediated by defective fork protection at stalled replication forks
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Angelica Barreto-Galvez, Mrunmai Niljikar, Julia Gagliardi, Ranran Zhang, Vasudha Kumar, Aastha Juruwala, Archana Pradeep, Anam Shaikh, Priyanka Tiwari, Kritika Sharma, Jeannine Gerhardt, Jian Cao, Keisuke Kataoka, Adam Durbin, Jun Qi, B. Hilda Ye, and Advaitha Madireddy
- Subjects
Article - Abstract
Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a model to study EP300 deficiency and an p300-selective PROTAC degrader, degrader as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in EP300-deficient cells. We demonstrate that the DNA replication dynamics of EP300-mutated cells ATLL cells recapitulate features of BRCA-deficient cancers. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress and defective replication fork restart results in persistent genomic instability that underlie aggressive chemo-resistant tumorigenesis in humans.
- Published
- 2023
6. The Cause and Consequence of Replication Stress in Adult T-Cell Leukemia
- Author
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Barreto-Galvez, Angelica, primary, Madireddy, Advaitha, additional, Ye, B. Hilda, additional, Gagliardi, Julia, additional, Juwarwala, Aastha, additional, Pradeep, Archana, additional, and Niljikar, Mrunmai, additional
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- 2022
- Full Text
- View/download PDF
7. Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites
- Author
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Ruth B De-Paula, Advaitha Madireddy, Shyam Twayana, William C. Drosopoulos, Settapong T. Kosiyatrakul, Albino Bacolla, John A. Tainer, Eric E. Bouhassira, Angelica Barreto-Galvez, and Carl L. Schildkraut
- Subjects
DNA Replication ,DNA Repair ,DNA polymerase ,Population ,SNP ,DNA-Directed DNA Polymerase ,replication fork pause ,Genomic Instability ,Cell Line ,chemistry.chemical_compound ,Proliferating Cell Nuclear Antigen ,Chromosome instability ,non-B DNA ,Humans ,education ,Gene ,Polymerase ,DNA Polymerase III ,Genetics ,education.field_of_study ,Multidisciplinary ,biology ,Chromosome Fragile Sites ,Chromosome Fragility ,Chromosomal fragile site ,DNA replication ,Genetic Variation ,DNA ,Cell Biology ,Biological Sciences ,chemistry ,biology.protein ,polymerase eta ,common fragile sites ,DNA Damage - Abstract
Significance Common fragile sites (CFSs) are normal loci that are genetically unstable under normal and oncogenic replication stress. Pol eta has been proposed to play a key role in CFS replication. Here, we show that in the absence of Pol eta, replication at five specific CFS loci is perturbed, with fork pausing observed at several sites. Sequence analysis showed that certain pause sites are associated with the presence of non-B DNA motifs, while others are not. Importantly, pause sites are located within regions of increased genetic variation in healthy human populations that could be attributed to Pol eta activity. Our data unveil a role for Pol eta in overcoming replication stress, reducing DNA breakage, and promoting genetic variation at CFSs., Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.
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- 2021
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8. The Cause and Consequence of Replication Stress in Adult T-Cell Leukemia
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Angelica Barreto-Galvez, Advaitha Madireddy, B. Hilda Ye, Julia Gagliardi, Aastha Juwarwala, Archana Pradeep, and Mrunmai Niljikar
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
- Full Text
- View/download PDF
9. Functional mapping of PHF6 complexes in chromatin remodeling, replication dynamics, and DNA repair
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Silvia Alvarez, Ana C. da Silva Almeida, Robert Albero, Mayukh Biswas, Angelica Barreto-Galvez, Thomas S. Gunning, Anam Shaikh, Tomas Aparicio, Agnieszka Wendorff, Erich Piovan, Pieter Van Vlierberghe, Steven Gygi, Jean Gautier, Advaitha Madireddy, and Adolfo A. Ferrando
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Leukemia ,DNA Repair ,MUTATIONS ,PROTEINS ,Immunology ,Biology and Life Sciences ,RECOMBINATION ,Cell Biology ,Hematology ,Chromatin Assembly and Disassembly ,Biochemistry ,Chromatin ,Nucleosomes ,FRAGILE SITES ,Repressor Proteins ,INSIGHTS ,HEMATOPOIETIC STEM ,FANCONI-ANEMIA ,Medicine and Health Sciences ,FAILURE ,Humans ,DAMAGE RESPONSE ,KAP-1 PHOSPHORYLATION - Abstract
The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes, including nucleosome remodeling and deacetylase, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, after DNA damage, PHF6 localizes to sites of DNA injury, and its loss impairs the resolution of DNA breaks, with consequent accumulation of single- and double-strand DNA lesions. Native chromatin immunoprecipitation sequencing analyses show that PHF6 specifically associates with difficult-to-replicate heterochromatin at satellite DNA regions enriched in histone H3 lysine 9 trimethyl marks, and single-molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling hematopoietic stem cell homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics, and DNA repair.
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- 2021
10. High burden of clonal hematopoiesis in first responders exposed to the World Trade Center disaster
- Author
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Sakshi Jasra, Orsi Giricz, Rachel Zeig-Owens, Kith Pradhan, David G. Goldfarb, Angelica Barreto-Galvez, Alexander J. Silver, Jiahao Chen, Srabani Sahu, Shanisha Gordon-Mitchell, Gaurav S. Choudhary, Srinivas Aluri, Tushar D. Bhagat, Aditi Shastri, Cosmin A. Bejan, Shannon S. Stockton, Travis P. Spaulding, Victor Thiruthuvanathan, Hiroki Goto, Jeannine Gerhardt, Syed Hissam Haider, Arul Veerappan, Matthias Bartenstein, George Nwankwo, Ola Landgren, Michael D. Weiden, Jacqueline Lekostaj, Ryan Bender, Frederick Fletcher, Lee Greenberger, Benjamin L. Ebert, Ulrich Steidl, Britta Will, Anna Nolan, Advaitha Madireddy, Michael R. Savona, David J. Prezant, and Amit Verma
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Disasters ,Mice ,Emergency Responders ,Animals ,Humans ,Dust ,General Medicine ,Clonal Hematopoiesis ,September 11 Terrorist Attacks ,General Biochemistry, Genetics and Molecular Biology ,Article - Abstract
The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64-6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population.
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- 2019
11. Understanding the Mechanisms Driving Genomic Instability in Adult T-Cell Leukemia/Lymphoma
- Author
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Madireddy, Advaitha, primary, Barreto-Galvez, Angelica, additional, and Ye, B. Hilda, additional
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- 2019
- Full Text
- View/download PDF
12. High Burden of Clonal Hematopoiesis in First Responders Exposed to the World Trade Center Disaster
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Jasra, Sakshi, primary, Giricz, Orsi, additional, Zeig-Owens, Rachel, additional, Goldfarb, David, additional, Barreto-Galvez, Angelica, additional, Pradhan, Kith, additional, Chen, Jiahao, additional, Choudhary, Gaurav S, additional, Aluri, Srinivas, additional, Bhagat, Tushar D, additional, Shastri, Aditi, additional, Thiruthuvanathan, Victor, additional, Goto, Hiroki, additional, Gerhardt, Jeannine, additional, Gordon, Shanisha, additional, Veerappan, Arul, additional, Haider, Syed Hissam, additional, Bartenstein, Matthias, additional, Nwankwo, George, additional, Landgren, Ola, additional, Weiden, Michael, additional, Fletcher, Frederick, additional, Greenberger, Lee, additional, Ebert, Benjamin L., additional, Steidl, Ulrich G., additional, Will, Britta, additional, Nolan, Anna, additional, Prezant, David, additional, Madireddy, Advaitha, additional, and Verma, Amit, additional
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- 2019
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- View/download PDF
13. High Burden of Clonal Hematopoiesis in First Responders Exposed to the World Trade Center Disaster
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Gaurav Choudhary, Michael D. Weiden, Angelica Barreto-Galvez, Anna Nolan, Amit Verma, Sakshi Jasra, Advaitha Madireddy, David J. Prezant, Srinivas Aluri, Victor Thiruthuvanathan, Syed Hissam Haider, Arul Veerappan, Benjamin L. Ebert, David G. Goldfarb, Shanisha Gordon, Jeannine Gerhardt, Lee M. Greenberger, Jiahao Chen, Orsolya Giricz, Britta Will, Ola Landgren, Rachel Zeig-Owens, Matthias Bartenstein, Hiroki Goto, Frederick A. Fletcher, George Nwankwo, Kith Pradhan, Tushar D. Bhagat, Aditi Shastri, and Ulrich Steidl
- Subjects
Oropharyngeal disorders ,File (record) ,business.industry ,Immunology ,Clonal hematopoiesis ,World trade center ,Cell Biology ,Hematology ,Hematologic Neoplasms ,Environmental exposure ,Biochemistry ,Medicine ,Idh2 gene ,business ,Personal Integrity ,Demography - Abstract
Introduction The World Trade Center (WTC) disaster exposed first responders to high levels of aerosolized carcinogens (Lioy et. al. Env. Health Perspect 2002). Clonal hematopoiesis is associated with exposure to smoking and genotoxic stimuli (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM 2015). We sought to determine its incidence in WTC-exposed first responders. We also assessed the effect of WTC particulate matter (WTC-PM) on genome integrity in vitro, and in murine studies. Methods Deep targeted sequencing was performed on blood collected from 481 first responders (429 WTC-exposed firefighters, 52 WTC-exposed emergency medical service workers) and 52 non-exposed first responders. Samples were analyzed for 237 genes mutated in hematologic malignancies and interpreted using reference databases. Non synonymous somatic mutations were annotated and analyzed. Results In the WTC-exposed cohort, 57 individuals with 66 somatic mutations of expected pathogenic potential were identified (overall prevalence 11.9%). In the non-exposed cohort, only one pathogenic mutation was found in the IDH2 gene (overall prevalence 1.9%). There was a strong association between increasing age and prevalence of mutations in the WTC-exposed cohort (Fig 1A). DNMT3A (16/66), TET2 (7/66), SF3B1 and SRSF2 (3/66 each) were the most common genes identified in the WTC-exposed cohort (Fig 1B). Median VAF was 12% and missense mutations were most frequent alteration. Aging, smoking, DNA repair and alkylating agent exposure related mutational signatures were observed with a cytosine to thymine (C→T) transition being most common. Next, we assessed the effect of WTC-PM on genome integrity and replication in vitro. WTC-PM that was collected in the first three days after 9/11 was used in concentrations mimicking exposure levels. Lymphocytes exposed to WTC-PM demonstrated a significant increase in phosphorylated H2AX foci accumulation, suggesting a DNA damage response (Fig 2). Since common fragile sites (CFSs) detect basal levels of stress in the cell, and activate DNA damage response (DDR), we profiled DNA replication dynamics at CFS-FRA16D at very high resolution using the single molecule analysis of replicated DNA (SMARD) assay. Treatment with WTC-PM significantly altered replication at two common fragile sites (regions 1 and 2 of FRA16D, Fig 3A) with replication pausing being observed at multiple sites (Fig 3B-I, white rectangles). Striking increase in replication initiation was seen, characterized as dormant origins activated to rescue replication pausing (Fig 3E, J). These alterations were accompanied by a corresponding increase in replication speed, conditions that lead to DNA replication errors and mutagenesis (Fig 3F, K). Next, we treated mice with WTC-PM via the oropharyngeal route to mimic first responder exposures, and then harvested and analyzed their bone marrow compartments. Significant expansion of hematopoietic stem cells (Kit+, Sca1+, Lineage-ve, KSL) was seen in WTC-PM treated mice (Fig 4A,B). Whole genome sequencing of sorted stem cells showed a significant increase in non-synonymous SNPs, deletions and indels in the WTC-PM treated samples when compared to control (Fig 4C-E). These genomic alterations were found to occur at low VAF throughout the whole genome, demonstrating widespread genotoxic effects of WTC-PM on hematopoietic stem cells in vivo (Fig 4F). Discussion We report a high burden of mutations in 11.9% (57/481) WTC-exposed first responders compared to the non-exposed cohort (1.9%, 1/52). The frequency of the somatic mutations was many fold higher than in previous studies (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM, 2015). In the 50-59 year age group, 10% of WTC-exposed individuals carried somatic mutations, compared to the frequency of 2.5% reported by Jaiswal et. al. for the same age group. Despite deeper sequencing performed in our study, the median VAF in our study was 12%, indicating that the difference in technique did not bias our study towards increased detection of small, subclinical clones when compared to previous studies. Furthermore, we demonstrate that WTC-PM can perturb DNA replication and increased genomic instability in vivo, potentially leading to higher burden of clonal hematopoiesis in WTC-exposed first responders. These results demonstrate adverse environmental exposures can be associated with a high rate of clonal hematopoiesis. Disclosures Landgren: Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Fletcher:Genoptix/Neogenomics: Employment. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Steidl:GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Will:Novartis Pharmaceuticals: Research Funding. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.
- Published
- 2019
- Full Text
- View/download PDF
14. Understanding the Mechanisms Driving Genomic Instability in Adult T-Cell Leukemia/Lymphoma
- Author
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Angelica Barreto-Galvez, Advaitha Madireddy, and B. Hilda Ye
- Subjects
Genome instability ,Immunology ,Cancer ,Cell Biology ,Hematology ,Biology ,medicine.disease ,biology.organism_classification ,Biochemistry ,Chemotherapy regimen ,Adult T-cell leukemia/lymphoma ,Lymphoma ,chemistry.chemical_compound ,chemistry ,immune system diseases ,hemic and lymphatic diseases ,Human T-lymphotropic virus 1 ,Host organism ,Cancer research ,medicine ,DNA - Abstract
Adult T-cell Leukemia/Lymphoma (ATLL) is a T-cell malignancy that results from infection by the retrovirus, human T cell lymphotropic virus-1 (HTLV-1). ATLL is endemic to Japan, the Caribbean regions and Latin America. Despite the rare occurrence of the disease, ATLL is a very aggressive malignancy with limited treatment options. Recent studies show that the ATLL patients diagnosed in North America (NA-ATLL), who are largely from the Caribbean region, have extremely poor prognosis as compared to the Japanese ATLL (J-ATLL) patients. A better understanding of the molecular pathogenesis of NA-ATLL is critical to identifying effective treatment measures for these patients. It has been previously shown that genomic instability including extensive chromosomal variations can be frequently found in ATLLs. However, the underlying mechanisms leading to this instability are unclear. Analysis of the mechanism of HTLV-1 action has revealed that the virus, in addition to hijacking the host cell machinery, disrupts DNA repair mechanisms and cell division processes. While the disruption of repair mechanisms could be held accountable for the accumulation of damage in ATLL cells, the precise nature of this disruption has not been fully understood. In proliferating cells, damage to the DNA occurs or is primarily recognized during DNA replication. This indicates that defective DNA replication could be an important factor driving genomic instability in ATLL cells. Possible involvement of replicative defects in the etiology of ATLL is further strengthened by the fact that genomic instability in ATLL cells has been shown to occur at difficult to replicate genomic regions referred to as common fragile sites. Here, using a powerful locus specific approach called the single molecule analysis of replicated DNA (SMARD), we show that perturbed DNA replication is an inherent component of disease manifestation in ATLL patients. Furthermore, our preliminary findings suggest that these changes in DNA replication can be largely attributed to the EP300 inactivating mutations often found among NA-ATLL patients. This study will help elucidate the molecular mechanisms contributing to the marked chemo-resistant feature of NA-ATLL patients, as compared to J-ATLL patients. In addition to increasing our understanding of the mechanisms contributing to ATLL in general, the results from this study may inform new and mechanism-based treatment paradigms that target the replicative defects of this unique disease. Disclosures No relevant conflicts of interest to declare.
- Published
- 2019
- Full Text
- View/download PDF
15. Acetyl transferase EP300 deficiency leads to chronic replication stress mediated by defective fork protection at stalled replication forks.
- Author
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Barreto-Galvez A, Niljikar M, Gagliardi J, Zhang R, Kumar V, Juruwala A, Pradeep A, Shaikh A, Tiwari P, Sharma K, Gerhardt J, Cao J, Kataoka K, Durbin A, Qi J, Ye BH, and Madireddy A
- Abstract
Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a model to study EP300 deficiency and an p300-selective PROTAC degrader, degrader as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in EP300-deficient cells. We demonstrate that the DNA replication dynamics of EP300-mutated cells ATLL cells recapitulate features of BRCA-deficient cancers. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress and defective replication fork restart results in persistent genomic instability that underlie aggressive chemo-resistant tumorigenesis in humans.
- Published
- 2023
- Full Text
- View/download PDF
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