55 results on '"Claudia M. Nicolae"'
Search Results
2. Supplementary Figure S8. WEE1 inhibitor in combination with IR inhibits cellular proliferation of normal and tumor cells. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
(A) Quantification of bands from Western gel shown in Main Figure 5D using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (B-E) Kinetic analysis of WT MEFs (B) as well as shScrambled D54 (C), HCT116 (D), and SCC61 (E) proliferation in vitro were measured over time in response to WEE1 inhibitor MK1775 {plus minus} IR (6 Gy) using the IncuCyte live cell imaging system. In vitro growth curve data are representative of at least three experiments, each with n = 3 per group. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.
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- 2023
3. Supplementary Figure S4. STING is upstream of CDKN1A signaling, but only has partial control of CDKN1A. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
(A-D) Quantification of bands from Western gels shown in Main Figures 3H (A), 3I (B), 3J (C), and 3K (D) using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (E) Western analysis of lysates harvested from HEK293 cells that were transiently transfected with either an empty vector of recombinant STING at 48 hours following exposure to 3Gy IR show that the protein levels of p21 and cyclin D1 were restored following overexpression of STING in HEK293 cells. (F) Western analysis of lysates harvested from siCDKN1A HCT116 cells at 48 hours following exposure to increasing dose of IR demonstrate that depletion of p21 did not affect STING expression, and only affected proteins that it regulates downstream of the pathway. (G) Western analysis of lysates harvested from MEFs that were transiently transfected with siRNA targeting STING and/or p21 at 48 hours reveal that depletion of STING led to down-regulation of p21, but not vice versa. Quantified bands are shown right next to each Western blot panels. (H-J) Kinetic analysis of STING-depleted human tumor cell lines D54 (H), HCT116 (I), and SCC61 (J) that were transiently transfected with siRNA targeting p21. Proliferation of nuclei-stained cells were measured in vitro over time using the IncuCyte live cell imaging system. The number of nuclei-stained cells were calculated using the Incucyte software and data are representative of three experiments, each with n = 3 per group. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.
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- 2023
4. Supplementary Figure S6. Ionizing radiation induces micronuclei formation in both WT and STING-/- MEFs. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
WT and STING-/- MEFs examined by cytokinesis-block micronucleus (CBMN) assay by treatment with IR, then cytochalasin at 24 hours and then fixed and stained for DNA and mitochondrial/cytoplasmic compartments using Draq5 and MitoTracker Red at 48 hours. Scale bar, 10 µm.
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- 2023
5. Supplementary Figure S7. The absence of STING (or p53 or p21) leads to increase in BUB1 and MAD2L1 expression. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
(A-C) BrdU/PI cell cycle profile of MEFs (A), p53-/- HCT116 (B), and CDKN1A-depleted HCT116 cells (C) at 24 hours post-IR. Flow cytometry was used to measure the percent cell population in G1, S, and G2/M phase of single cells stained with FITC-conjugated anti-BrdU (y-axis) and PI (x-axis). (D-F) Western analyses of lysates from WT and STING-/- immortalized MEFs (D), p53-negative HCT116 (E), and siCDKN1A HCT116 (F) probed for mitotic proteins BUB1 and MAD2L1 at 48 hours post-exposure to increasing IR doses. Quantified bands are shown below each of their respective Western blot panels. (G) ChIP analyses reveal that in the absence of either p21 or STING, E2F4 does not bind to the promoter region of BUB1 and MAD2L1 in HCT116 tumor cells. Data are representative of at least two experiments. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.
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- 2023
6. Data from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
Given the integral role of stimulator of interferon genes (STING, TMEM173) in the innate immune response, its loss or impairment in cancer is thought to primarily affect antitumor immunity. Here we demonstrate a role for STING in the maintenance of cellular homeostasis through regulation of the cell cycle. Depletion of STING in human and murine cancer cells and tumors resulted in increased proliferation compared with wild-type controls. Microarray analysis revealed genes involved in cell-cycle regulation are differentially expressed in STINGko compared with WT MEFs. STING-mediated regulation of the cell cycle converged on NFκB- and p53-driven activation of p21. The absence of STING led to premature activation of cyclin-dependent kinase 1 (CDK1), early onset to S-phase and mitosis, and increased chromosome instability, which was enhanced by ionizing radiation. These results suggest a pivotal role for STING in maintaining cellular homeostasis and response to genotoxic stress.Significance:These findings provide clear mechanistic understanding of the role of STING in cell-cycle regulation, which may be exploited in cancer therapy because most normal cells express STING, while many tumor cells do not.See related commentary by Gius and Zhu, p. 1295
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- 2023
7. Supplementary Figure S2. STING-dependent regulation of proliferation is associated with perturbations of cell cycle. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
- Author
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
Cell cycle profile of pulse EdU-labeled (A) and chase EdU-labeled (B) WT and STING-/- MEFs at different time points post-IR treatment. Flow cytometry was used to measure the percent cell population in G1, S, and G2/M phase of single cells stained with AlexaFluor 647-conjugated anti-EdU (y-axis) and PI (x-axis).
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- 2023
8. Supplementary Figure S1. shRNA constructs targeting STING have varying effects on different tumor cell lines. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
- Author
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
Western blot analysis of multiple stable D54 (A), HCT116 (B), and MC-38 (C) cell lines expressing short hairpin RNAs targeting STING or scrambled control (SHCOO2). Quantified bands are shown below each of their respective Western blot panels.
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- 2023
9. Supplementary Figure S5. Quantification of Western blot bands using Image J. from STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
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Ralph R. Weichselbaum, Nikolai N. Khodarev, George-Lucian Moldovan, Stephen J. Kron, Renate Parry, Ainhoa Arina, Michael J. Bolt, Xiaona Huang, Claudia M. Nicolae, Akash D. Parekh, Stephen Mallon, Ryan C. Widau, and Diana Rose E. Ranoa
- Abstract
The pixel units obtained for each protein band from Western gels shown in Main Figures 4B (A), 4C (B), 4D (C), and 4E (D) was normalized to the pixel units calculated from their respective b-Actin loading control. For phosphorylated proteins, we then quantified the fraction of phosphorylated proteins to the absolute total amount of protein (total protein + phosphorylated protein). For dose-dependent and stimulation assays, the variables tested were normalized to their respective wild-type unstimulated controls.
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- 2023
10. Supplementary Figures 1 - 11 from PARI Overexpression Promotes Genomic Instability and Pancreatic Tumorigenesis
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George-Lucian Moldovan, Alan D. D'Andrea, Alec C. Kimmelman, Claudia M. Nicolae, Eunmi Park, Donniphat Dejsuphong, and Kevin W. O'Connor
- Abstract
PDF file - 2404K, Supplementary Figures 1-11; Supplementary Figure 1: Representative micrographs showing a decrease in IR-induced RAD51 foci in PARI-overexpressing 8988T and CAPAN2 cells. Supplementary Figure 2: PARI-overexpressing pancreatic cancer cell line 8988T is hypersensitive to PARP inhibitor ABT-888. Supplementary Figure 3: SupF-based mutagenesis assay showing increased point mutations in 293T cells overexpressing Myc-PARI at high levels. Supplementary Figure 4: Analysis of chromosome morphology in metaphase spreads of 8988T cells, showing that PARI knockdown reduces chromosomal aberrations following exposure to 20ng/ml MMC. Supplementary Figure 5: PARI depletion reduces viability of PARI-overexpressing cells (CAPAN2), but does not affect the cells that do not overexpress it (CAPAN1, HeLa, 293T). Supplementary Figure 6: PARI knockdown leads to a significant increase in the S-phase fraction. Supplementary Figure 7: FACS analysis of 8988T cells treated with HU, followed by release in fresh media. Supplementary Figure 8: PARI depletion in CAPAN2 (pancreatic cancer cells overexpressing PARI) results in HU sensitivity. Supplementary Figure 9: Depletion of PARI by shRNA decreases cellular proliferation when grown in the presence of 20μg/ml doxycyline. Supplementary Figure 10: H&E stain of paraffin-embedded slides showing increased necrosis in xenograft tumors originating from PARI-depleted 8988T cells. Supplementary Figure 11: Model of PARI-dependent DNA repair regulation in pancreatic cancer cells.
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- 2023
11. Supplementary Methods and Figure Legends from PARI Overexpression Promotes Genomic Instability and Pancreatic Tumorigenesis
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George-Lucian Moldovan, Alan D. D'Andrea, Alec C. Kimmelman, Claudia M. Nicolae, Eunmi Park, Donniphat Dejsuphong, and Kevin W. O'Connor
- Abstract
PDF file - 62K, Supplemental Materials and Methods, including: Antibodies, Gene knockdown by siRNA, and Histology.
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- 2023
12. Mono-ADP-ribosylation by PARP10 and PARP14 in genome stability
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Ashna Dhoonmoon and Claudia M Nicolae
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General Medicine - Abstract
ADP-ribosylation is a post-translational modification involved in a variety of processes including DNA damage repair, transcriptional regulation, and cellular proliferation. Depending on the number of ADP moieties transferred to target proteins, ADP-ribosylation can be classified either as mono-ADP-ribosylation (MARylation) or poly-ADP-ribosylation (PARylation). This post-translational modification is catalyzed by enzymes known as ADP-ribosyltransferases (ARTs), which include the poly (ADP-ribose)-polymerase (PARP) superfamily of proteins. Certain members of the PARP family including PARP1 and PARP2 have been extensively studied and assessed as therapeutic targets. However, the other members of the PARP family of protein are not as well studied but have gained attention in recent years given findings suggesting their roles in an increasing number of cellular processes. Among these other members are PARP10 and PARP14, which have gradually emerged as key players in maintenance of genomic stability and carcinogenesis. PARP10 and PARP14 catalyze the transfer of a single ADP moiety to target proteins. Here, we summarize the current knowledge on MARylation in DNA repair and cancer, focusing on PARP10 and PARP14. We highlight the roles of PARP10 and PARP14 in cancer progression and response to chemotherapeutics and briefly discuss currently known PARP10 and PARP14 inhibitors.
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- 2023
13. Complementary CRISPR genome-wide genetic screens in PARP10-knockout and overexpressing cells identify synthetic interactions for PARP10-mediated cellular survival
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Jude B. Khatib, Emily M. Schleicher, Lindsey M. Jackson, Ashna Dhoonmoon, George-Lucian Moldovan, and Claudia M. Nicolae
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ADP Ribose Transferases ,Oncology ,Neoplasms ,Proto-Oncogene Proteins ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,DNA ,Poly(ADP-ribose) Polymerases ,Precision Medicine - Abstract
PARP10 is a mono-ADP-ribosyltransferase with multiple cellular functions, including proliferation, apoptosis, metabolism and DNA repair. PARP10 is overexpressed in a significant proportion of tumors, particularly breast and ovarian cancers. Identifying genetic susceptibilities based on PARP10 expression levels is thus potentially relevant for finding new targets for precision oncology. Here, we performed a series of CRISPR genome-wide loss-of-function screens in isogenic control and PARP10-overexpressing or PARP10-knockout cell lines, to identify genetic determinants of PARP10-mediated cellular survival. We found that PARP10-overexpressing cells rely on multiple DNA repair genes for survival, including ATM, the master regulator of the DNA damage checkpoint. Moreover, we show that PARP10 impacts the recruitment of ATM to nascent DNA upon replication stress. Finally, we identify the CDK2-Cyclin E1 complex as essential for proliferation of PARP10-knockout cells. Our work identifies a network of functionally relevant PARP10 synthetic interactions, and reveals a set of factors which can potentially be targeted in personalized cancer therapy.
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- 2022
14. The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells
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Emily M. Schleicher, Ashna Dhoonmoon, Lindsey M. Jackson, Jude B. Khatib, Claudia M. Nicolae, and George-Lucian Moldovan
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Cancer Research ,Molecular Biology - Abstract
Maintenance of replication fork stability is essential for genome preservation. Stalled replication forks can be reversed by translocases such as SMARCAL1, and unless protected through the activity of the BRCA pathway, are subsequently subjected to nucleolytic degradation. The ATM and ATR kinases are master regulators of the DNA damage response. ATM activation upon DNA damage is mediated by the acetyltransferase TIP60. Here, we show that the TIP60-ATM pathway promotes replication fork reversal by recruiting SMARCAL1 to stalled forks. This enables fork degradation in BRCA-deficient cells. We also show that this ATM activity is not shared by ATR. Moreover, we performed a series of genome-wide CRISPR knockout genetic screens to identify genetic determinants of the cellular sensitivity to ATM inhibition in wildtype and BRCA2-knockout cells, and validated the top hits from multiple screens. We provide a valuable list of common genes which regulate the response to multiple ATM inhibitors. Importantly, we identify a differential response of wildtype and BRCA2-deficient cells to these inhibitors. In BRCA2-knockout cells, DNA repair genes (including RAD17, MDC1, and USP28) were essential for survival upon ATM inhibitor treatment, which was not the case in wild-type cells. These findings may eventually help guide the way for rational deployment of ATM inhibitors in the clinic.
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- 2022
15. Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly through PCNA-dependent CAF-1 recycling
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Tanay Thakar, Ashna Dhoonmoon, Joshua Straka, Emily M. Schleicher, Claudia M. Nicolae, and George-Lucian Moldovan
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Chromatin Assembly Factor-1 ,Multidisciplinary ,Proliferating Cell Nuclear Antigen ,General Physics and Astronomy ,DNA, Single-Stranded ,Recycling ,General Chemistry ,Chromatin Assembly and Disassembly ,General Biochemistry, Genetics and Molecular Biology ,Nucleosomes - Abstract
The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and underlies chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA-deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that lagging strand ssDNA gaps interfere with the ASF1-CAF-1 nucleosome assembly pathway, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to defects in its recycling during replication stress. This CAF-1 recycling defect is caused by lagging strand gaps which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores CAF-1-dependent fork stability. We further show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells. We thus define lagging strand gap suppression and nucleosome assembly as critical enablers of BRCA-mediated fork stability.
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- 2022
16. FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links
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Robert M. Brosh, Arindam Datta, Marina A. Bellani, Sanket Awate, Christopher A. Dunn, Joshua A. Sommers, Michael M. Seidman, Sumeet Nayak, George Lucian Moldovan, Claudia M. Nicolae, Olivia Yang, and Sharon B. Cantor
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Genome instability ,DNA Repair ,DNA damage ,DNA repair ,Mitomycin ,RAD51 ,Genome Integrity, Repair and Replication ,Genomic Instability ,Gene Knockout Techniques ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Chromosomal Instability ,Genetics ,Humans ,BRIP1 Gene ,DNA Breaks, Double-Stranded ,Histone Chaperones ,030304 developmental biology ,0303 health sciences ,biology ,BRCA1 Protein ,Recombinational DNA Repair ,Helicase ,Fanconi Anemia Complementation Group Proteins ,Cell biology ,DNA-Binding Proteins ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Rad51 Recombinase ,Homologous recombination ,RNA Helicases ,DNA ,DNA Damage ,HeLa Cells - Abstract
FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.
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- 2020
17. Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly
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Claudia M. Nicolae, Wendy Leung, Tanay Thakar, Binghui Shen, Kristen E. Clements, George Lucian Moldovan, and Anja Katrin Bielinsky
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DNA Replication ,0301 basic medicine ,Genome instability ,DNA Repair ,DNA polymerase ,Science ,Fluorescent Antibody Technique ,General Physics and Astronomy ,Article ,Genomic Instability ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Ubiquitin ,Cell Line, Tumor ,Proliferating Cell Nuclear Antigen ,Humans ,Nucleosome ,lcsh:Science ,Multidisciplinary ,biology ,Okazaki fragments ,DNA damage and repair ,HEK 293 cells ,Ubiquitination ,Stalled forks ,DNA ,General Chemistry ,Chromatin Assembly and Disassembly ,Cell biology ,Proliferating cell nuclear antigen ,HEK293 Cells ,030104 developmental biology ,chemistry ,biology.protein ,lcsh:Q ,Comet Assay ,030217 neurology & neurosurgery ,DNA Damage ,HeLa Cells ,Protein Binding - Abstract
Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors., PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.
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- 2020
18. Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling
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Tanay Thakar, Joshua Straka, Claudia M. Nicolae, and George Lucian Moldovan
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Genome instability ,chemistry.chemical_compound ,Histone ,biology ,Nucleosome assembly ,Chemistry ,Fork (system call) ,biology.protein ,DNA ,Proliferating cell nuclear antigen ,Chromatin ,CAF-1 ,Cell biology - Abstract
The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and is associated with chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that ssDNA gaps on the lagging strand interfere with the ASF1-CAF-1 pathway of nucleosome assembly, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to its sequestration at inactive replication factories during replication stress. This CAF-1 recycling defect is caused by the accumulation of Polα-dependent lagging strand gaps, which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores fork stability in a CAF-1-dependent manner. We also show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells upon CAF-1 loss. We thus define nucleosome assembly as a critical determinant of BRCA-mediated fork stability. We further reveal lagging strand ssDNA gaps as drivers of fork degradation in BRCA-deficient cells, which operate by inhibiting PCNA unloading and CAF-1-dependent nucleosome assembly.
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- 2021
19. WRN helicase safeguards deprotected replication forks in BRCA2-mutated cancer cells
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Sanket Awate, Kajal Biswas, Joshua A. Sommers, Shyam K. Sharan, Tanay Thakar, Claudia M. Nicolae, Robert M. Brosh, Arindam Datta, Robert H. Shoemaker, Haley Thompson, and George Lucian Moldovan
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DNA Replication ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,DNA damage ,Science ,Poly ADP ribose polymerase ,Poly (ADP-Ribose) Polymerase-1 ,Mice, Nude ,General Physics and Astronomy ,Poly(ADP-ribose) Polymerase Inhibitors ,Genomic Instability ,General Biochemistry, Genetics and Molecular Biology ,Article ,Mice ,Cell Line, Tumor ,Neoplasms ,Chromosome instability ,Animals ,Cancer genetics ,Polymerase ,BRCA2 Protein ,MRE11 Homologue Protein ,Nuclease ,Multidisciplinary ,biology ,Chemistry ,DNA Helicases ,Helicase ,nutritional and metabolic diseases ,General Chemistry ,Stalled forks ,Cell biology ,Chromatin ,enzymes and coenzymes (carbohydrates) ,Cancer cell ,biology.protein ,Heterografts ,Female ,DNA Damage - Abstract
The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. However, the molecular mechanism(s) whereby unprotected forks are stabilized remains to be fully characterized. Here, we demonstrate that WRN helicase ensures efficient restart and limits excessive degradation of stalled forks in BRCA2-deficient cancer cells. In vitro, WRN ATPase/helicase catalyzes fork restoration and curtails MRE11 nuclease activity on regressed forks. We show that WRN helicase inhibitor traps WRN on chromatin leading to rapid fork stalling and nucleolytic degradation of unprotected forks by MRE11, resulting in MUS81-dependent double-strand breaks, elevated non-homologous end-joining and chromosomal instability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. Furthermore, BRCA2-deficient xenograft tumors in mice exhibited increased DNA damage and growth inhibition when treated with WRN helicase inhibitor. This work provides mechanistic insight into stalled fork stabilization by WRN helicase when BRCA2 is deficient., The tumor suppressor BRCA2 protects stalled DNA replication forks from unrestrained degradation; however the mechanism whereby unprotected stalled forks are preserved and restarted has remained elusive. Here the authors show that the WRN helicase promotes stalled fork recovery and limits fork hyper-degradation in the absence of BRCA2 protection.
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- 2021
20. Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells
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Claudia M. Nicolae, George Lucian Moldovan, Jenna Thomforde, Zhongtao Zhang, Natalia Y. Tretyakova, Shaofei Ji, Marietta Y.W.T. Lee, Spandana Naldiga, and Ashis K. Basu
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DNA Replication ,0301 basic medicine ,DNA damage ,DNA polymerase ,DNA repair ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,Cytosine ,DNA Adducts ,Gene Knockout Techniques ,03 medical and health sciences ,chemistry.chemical_compound ,Plasmid ,Humans ,Molecular Biology ,reproductive and urinary physiology ,Polymerase ,DNA Polymerase III ,DNA Primers ,030102 biochemistry & molecular biology ,biology ,DNA replication ,DNA ,DNA Polymerase II ,Cell Biology ,Molecular biology ,Chromatin ,HEK293 Cells ,030104 developmental biology ,chemistry ,Mutation ,embryonic structures ,biology.protein ,Peptides - Abstract
DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.
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- 2019
21. PARI (PARPBP) suppresses replication stress-induced myeloid differentiation in leukemia cells
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Chunhua Song, Emily M. Schleicher, George Lucian Moldovan, Raghavendra Gowda, Michael J. O’Connor, Gavin P. Robertson, Sinisa Dovat, and Claudia M. Nicolae
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0301 basic medicine ,Cancer Research ,Gene knockdown ,Myeloid ,DNA damage ,Cell growth ,DNA repair ,Myeloid leukemia ,Biology ,medicine.disease ,Cell biology ,03 medical and health sciences ,Leukemia ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,hemic and lymphatic diseases ,030220 oncology & carcinogenesis ,Cancer cell ,Genetics ,medicine ,Molecular Biology - Abstract
Hyperproliferative cancer cells face increased replication stress, which can result in accumulation of DNA damage. As DNA damage can arrest proliferation, and, in the case of myeloid leukemia, induce differentiation of cancer cells, understanding the mechanisms that regulate the replication stress response is paramount. Here, we show that PARI, a replisome protein involved in regulating DNA repair and replication stress, suppresses differentiation of myeloid leukemia cells. We show that PARI is overexpressed in myeloid leukemia cells, and its knockdown reduces leukemia cell proliferation in vitro and in vivo in xenograft mouse models. PARI depletion enhances replication stress and DNA-damage accumulation, coupled with increased myeloid differentiation. Mechanistically, we show that PARI inhibits activation of the NF-κB pathway, which can initiate p21-mediated differentiation and proliferation arrest. Finally, we show that PARI expression negatively correlates with expression of differentiation markers in clinical myeloid leukemia samples, suggesting that targeting PARI may restore differentiation ability of leukemia cells and antagonize their proliferation.
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- 2019
22. Heterozygous RNF13 Gain-of-Function Variants Are Associated with Congenital Microcephaly, Epileptic Encephalopathy, Blindness, and Failure to Thrive
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Orly Elpeleg, Claudia M. Nicolae, Anna De Grassi, Jessica Bischetsrieder, Simon Edvardson, Giuseppe Punzi, Jennifer Burton, George Lucian Moldovan, Ciro Leonardo Pierri, Grace J. Noh, and Avraham Shaag
- Subjects
Male ,Models, Molecular ,0301 basic medicine ,Heterozygote ,Microcephaly ,Developmental Disabilities ,Ubiquitin-Protein Ligases ,Cell ,Apoptosis ,Biology ,Blindness ,03 medical and health sciences ,Epilepsy ,0302 clinical medicine ,Report ,Genetics ,medicine ,Humans ,Amino Acid Sequence ,Child ,Genetics (clinical) ,Endoplasmic reticulum ,Neurodegeneration ,Infant ,Endoplasmic Reticulum Stress ,medicine.disease ,Failure to Thrive ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Child, Preschool ,Gain of Function Mutation ,Failure to thrive ,Unfolded Protein Response ,Unfolded protein response ,medicine.symptom ,Spasms, Infantile ,030217 neurology & neurosurgery - Abstract
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates a stress response mechanism to clear out the unfolded proteins by either facilitating their re-folding or inducing their degradation. When this fails, an apoptotic cascade is initiated so that the affected cell is eliminated. IRE1α is a critical sensor of the unfolded-protein response, essential for initiating the apoptotic signaling. Here, we report an infantile neurodegenerative disorder associated with enhanced activation of IRE1α and increased apoptosis. Three unrelated affected individuals with congenital microcephaly, infantile epileptic encephalopathy, and profound developmental delay were found to carry heterozygous variants (c.932T>C [p.Leu311Ser] or c.935T>C [p.Leu312Pro]) in RNF13, which codes for an IRE1α-interacting protein. Structural modeling predicted that the variants, located on the surface of the protein, would not alter overall protein folding. Accordingly, the abundance of RNF13 and IRE1α was not altered in affected individuals’ cells. However, both IRE1α-mediated stress signaling and stress-induced apoptosis were increased in affected individuals’ cells. These results indicate that the RNF13 variants confer gain of function to the encoded protein and thereby lead to altered signaling of the ER stress response associated with severe neurodegeneration in infancy.
- Published
- 2019
23. RECON syndrome is a genome instability disorder caused by mutations in the DNA helicase RECQL1
- Author
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Bassam Abu-Libdeh, Satpal S. Jhujh, Srijita Dhar, Joshua A. Sommers, Arindam Datta, Gabriel M.C. Longo, Laura J. Grange, John J. Reynolds, Sophie L. Cooke, Gavin S. McNee, Robert Hollingworth, Beth L. Woodward, Anil N. Ganesh, Stephen J. Smerdon, Claudia M. Nicolae, Karina Durlacher-Betzer, Vered Molho-Pessach, Abdulsalam Abu-Libdeh, Vardiella Meiner, George-Lucian Moldovan, Vassilis Roukos, Tamar Harel, Robert M. Brosh, and Grant S. Stewart
- Subjects
DNA Replication ,RecQ Helicases ,Mutation ,Humans ,Breast Neoplasms ,Female ,Genetic Predisposition to Disease ,General Medicine ,Genomic Instability - Abstract
Despite being the first homolog of the bacterial RecQ helicase to be identified in humans, the function of RECQL1 remains poorly characterized. Furthermore, unlike other members of the human RECQ family of helicases, mutations in RECQL1 have not been associated with a genetic disease. Here, we identify 2 families with a genome instability disorder that we have named RECON (RECql ONe) syndrome, caused by biallelic mutations in the RECQL gene. The affected individuals had short stature, progeroid facial features, a hypoplastic nose, xeroderma, and skin photosensitivity and were homozygous for the same missense mutation in RECQL1 (p.Ala459Ser), located within its zinc binding domain. Biochemical analysis of the mutant RECQL1 protein revealed that the p.A459S missense mutation compromised its ATPase, helicase, and fork restoration activity, while its capacity to promote single-strand DNA annealing was largely unaffected. At the cellular level, this mutation in RECQL1 gave rise to a defect in the ability to repair DNA damage induced by exposure to topoisomerase poisons and a failure of DNA replication to progress efficiently in the presence of abortive topoisomerase lesions. Taken together, RECQL1 is the fourth member of the RecQ family of helicases to be associated with a human genome instability disorder.
- Published
- 2021
24. Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens
- Author
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Hong Gang Wang, Anchal Sharma, Anastasia Hale, Tanay Thakar, Xinwen Liang, Ashna Dhoonmoon, Claudia M. Nicolae, Nathanial J. Tolman, Kristen E. Clements, George Lucian Moldovan, Yuka Imamura Kawasawa, Emily M. Schleicher, and Subhajyoti De
- Subjects
0301 basic medicine ,endocrine system diseases ,Cancer therapy ,DNA Repair ,DNA repair ,DNA damage ,Science ,Ubiquitin-Protein Ligases ,RAD51 ,General Physics and Astronomy ,Poly(ADP-ribose) Polymerase Inhibitors ,Poly (ADP-Ribose) Polymerase Inhibitor ,Article ,Lysine Acetyltransferase 5 ,General Biochemistry, Genetics and Molecular Biology ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,Humans ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,skin and connective tissue diseases ,Homologous Recombination ,Polymerase ,BRCA2 Protein ,Multidisciplinary ,biology ,Chemistry ,Tumor Suppressor Proteins ,fungi ,DNA damage and repair ,food and beverages ,General Chemistry ,Ubiquitin ligase ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,Mad2 Proteins ,biology.protein ,Rad51 Recombinase ,Poly(ADP-ribose) Polymerases ,Tumor Suppressor p53-Binding Protein 1 ,Homologous recombination ,Biomarkers ,DNA Damage ,HeLa Cells - Abstract
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells., Mutations in the homologous recombination proteins BRCA1 and BRCA2 can sensitize cells to treatment with inhibitors of poly-ADP-ribose polymerase 1 (PARPi), but resistance to the treatment can occur. Here the authors by genome-wide CRISPR knockout and activation screens reveal novel pathways of PARPi resistance in BRCA2-deficient cells.
- Published
- 2020
25. Genome-wide CRISPR synthetic lethality screen identifies a role for the ADP-ribosyltransferase PARP14 in DNA replication dynamics controlled by ATR
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Kristen E. Clements, George Lucian Moldovan, Claudia M. Nicolae, Emily M. Schleicher, and Ashna Dhoonmoon
- Subjects
DNA Replication ,AcademicSubjects/SCI00010 ,DNA repair ,DNA damage ,Ataxia Telangiectasia Mutated Proteins ,Synthetic lethality ,Genome Integrity, Repair and Replication ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,Humans ,CRISPR ,030304 developmental biology ,0303 health sciences ,DNA replication ,Cell cycle ,Cell biology ,chemistry ,030220 oncology & carcinogenesis ,Checkpoint Kinase 1 ,biological phenomena, cell phenomena, and immunity ,CRISPR-Cas Systems ,Poly(ADP-ribose) Polymerases ,Synthetic Lethal Mutations ,DNA ,Genetic screen - Abstract
The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.
- Published
- 2020
26. PARP14 regulates cyclin D1 expression to promote cell-cycle progression
- Author
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George Lucian Moldovan, Tanay Thakar, Michael J. O’Connor, and Claudia M. Nicolae
- Subjects
0301 basic medicine ,Cancer Research ,Poly ADP ribose polymerase ,RNA Stability ,Regulator ,Biology ,Retinoblastoma Protein ,Article ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Cyclin D1 ,Genetics ,medicine ,Humans ,Luciferase ,RNA, Messenger ,RNA, Small Interfering ,Molecular Biology ,3' Untranslated Regions ,Polymerase ,Messenger RNA ,Retinoblastoma ,Cell Cycle ,medicine.disease ,G1 Phase Cell Cycle Checkpoints ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,biology.protein ,RNA Interference ,Signal transduction ,Poly(ADP-ribose) Polymerases ,E2F1 Transcription Factor - Abstract
Cyclin D1 is an essential regulator of the G1–S cell-cycle transition and is overexpressed in many cancers. Expression of cyclin D1 is under tight cellular regulation that is controlled by many signaling pathways. Here we report that PARP14, a member of the poly(ADP-ribose) polymerase (PARP) family, is a regulator of cyclin D1 expression. Depletion of PARP14 leads to decreased cyclin D1 protein levels. In cells with a functional retinoblastoma (RB) protein pathway, this results in G1 cell-cycle arrest and reduced proliferation. Mechanistically, we found that PARP14 controls cyclin D1 mRNA levels. Using luciferase assays, we show that PARP14 specifically regulates cyclin D1 3′UTR mRNA stability. Finally, we also provide evidence that G1 arrest in PARP14-depleted cells is dependent on an intact p53–p21 pathway. Our work uncovers a new role for PARP14 in promoting cell-cycle progression through both cyclin D1 and the p53 pathway.
- Published
- 2020
27. Dual genome-wide CRISPR knockout and CRISPR activation screens identify common mechanisms that regulate the resistance to multiple ATR inhibitors
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Claudia M. Nicolae, Emily M. Schleicher, Lindsey M. Jackson, Kristen E. Clements, George Lucian Moldovan, Ashna Dhoonmoon, and Coryn L. Stump
- Subjects
0303 health sciences ,DNA damage ,Regulator ,DNA replication ,Biology ,medicine.disease ,Genome ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,030220 oncology & carcinogenesis ,Cancer cell ,Ataxia-telangiectasia ,medicine ,CRISPR ,biological phenomena, cell phenomena, and immunity ,Gene ,030304 developmental biology - Abstract
The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication tract progression, and prevention of ATR inhibitor-induced apoptosis. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.
- Published
- 2020
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- View/download PDF
28. Genome-wide CRISPR synthetic lethality screen identifies a role for the ADP-ribosyltransferase PARP14 in replication fork stability controlled by ATR
- Author
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Ashna Dhoonmoon, Claudia M. Nicolae, Emily M. Schleicher, Kristen E. Clements, and George Lucian Moldovan
- Subjects
0303 health sciences ,DNA damage ,DNA repair ,Synthetic lethality ,Biology ,medicine.disease_cause ,Genome ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,030220 oncology & carcinogenesis ,medicine ,CRISPR ,biological phenomena, cell phenomena, and immunity ,Carcinogenesis ,030304 developmental biology ,Genetic screen - Abstract
The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of replication fork stability as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.
- Published
- 2020
- Full Text
- View/download PDF
29. Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens
- Author
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Yuka Imamura Kawasawa, Anastasia Hale, Claudia M. Nicolae, Xinwen Liang, Anchal Sharma, Kristen E. Clements, George Lucian Moldovan, Tanay Thakar, Hong Gang Wang, Nathanial J. Tolman, and Subhajyoti De
- Subjects
0303 health sciences ,biology ,Poly ADP ribose polymerase ,Histone acetyltransferase ,DNA Repair Pathway ,Double Strand Break Repair ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,030220 oncology & carcinogenesis ,PARP inhibitor ,biology.protein ,CRISPR ,Homologous recombination ,Polymerase ,030304 developmental biology - Abstract
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in the homologous recombination (HR) DNA repair pathway, such as those lacking BRCA2. In light of this, PARPi have been utilized in recent years to treat BRCA2-mutant tumors, with many patients deriving impressive clinical benefit. However, positive response to PARPi is not universal, even among patients with HR-deficient tumors. Here, we present the results of three genome-wide CRISPR knockout and activation screens which provide an unbiased look at genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we reveal that depletion of the histone acetyltransferase TIP60, a top hit from our screens, robustly reverses the PARPi sensitivity caused by BRCA2 deficiency. Mechanistically, we show that TIP60 depletion rewires double strand break repair in BRCA2-deficient cells by promoting 53BP1 binding to double strand breaks to suppress end resection. Our work provides a comprehensive set of putative biomarkers that serve to better understand and predict PARPi response, and identifies a novel pathway of PARPi resistance in BRCA2-deficient cells.
- Published
- 2019
- Full Text
- View/download PDF
30. PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly
- Author
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Tanay Thakar, Claudia M. Nicolae, Wendy Leung, Binghui Shen, Anja Katrin Bielinsky, Kristen E. Clements, and George Lucian Moldovan
- Subjects
Genome instability ,chemistry.chemical_compound ,Ubiquitin ,biology ,Okazaki fragments ,Cas9 ,Chemistry ,DNA polymerase ,biology.protein ,CRISPR ,DNA ,Cell biology ,Proliferating cell nuclear antigen - Abstract
Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing ubiquitination-deficient 293T and RPE1 cells generated through CRISPR/Cas9 genome editing, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-mediated but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork, and incomplete Okazaki fragment synthesis and maturation, thus interfering with efficient PCNA unloading by ATAD5 and subsequent nucleosomal deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and BRCA2 results in a synergistic increase in nascent DNA degradation and sensitivity to PARP-inhibitors. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.
- Published
- 2019
31. PARI (PARPBP) suppresses replication stress-induced myeloid differentiation in leukemia cells
- Author
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Claudia M, Nicolae, Michael J, O'Connor, Emily M, Schleicher, Chunhua, Song, Raghavendra, Gowda, Gavin, Robertson, Sinisa, Dovat, and George-Lucian, Moldovan
- Subjects
DNA-Binding Proteins ,Leukemia, Myeloid ,Gene Knockdown Techniques ,NF-kappa B ,Humans ,Cell Differentiation ,HL-60 Cells ,U937 Cells ,Cell Proliferation ,DNA Damage ,Protein Binding - Abstract
Hyperproliferative cancer cells face increased replication stress, which can result in accumulation of DNA damage. As DNA damage can arrest proliferation, and, in the case of myeloid leukemia, induce differentiation of cancer cells, understanding the mechanisms that regulate the replication stress response is paramount. Here, we show that PARI, a replisome protein involved in regulating DNA repair and replication stress, suppresses differentiation of myeloid leukemia cells. We show that PARI is overexpressed in myeloid leukemia cells, and its knockdown reduces leukemia cell proliferation in vitro and in vivo in xenograft mouse models. PARI depletion enhances replication stress and DNA-damage accumulation, coupled with increased myeloid differentiation. Mechanistically, we show that PARI inhibits activation of the NF-κB pathway, which can initiate p21-mediated differentiation and proliferation arrest. Finally, we show that PARI expression negatively correlates with expression of differentiation markers in clinical myeloid leukemia samples, suggesting that targeting PARI may restore differentiation ability of leukemia cells and antagonize their proliferation.
- Published
- 2018
32. Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors
- Author
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Ashna Dhoonmoon, Coryn L. Stump, Claudia M. Nicolae, Emily M. Schleicher, Lindsey M. Jackson, Kristen E. Clements, and George Lucian Moldovan
- Subjects
Genetic Screens ,Cancer Research ,Indoles ,Gene Identification and Analysis ,Cultured tumor cells ,Cancer Treatment ,Apoptosis ,Ataxia Telangiectasia Mutated Proteins ,QH426-470 ,Synthetic Genome Editing ,Biochemistry ,Genome Engineering ,0302 clinical medicine ,Transforming Growth Factor beta ,Neoplasms ,Medicine and Health Sciences ,CRISPR ,Genetics (clinical) ,Sulfonamides ,0303 health sciences ,Mediator Complex ,Cell Death ,Crispr ,Small interfering RNA ,Cell biology ,Nucleic acids ,Oncology ,Cell Processes ,Gene Knockdown Techniques ,Sulfoxides ,030220 oncology & carcinogenesis ,Cell lines ,Engineering and Technology ,Synthetic Biology ,biological phenomena, cell phenomena, and immunity ,Signal transduction ,Biological cultures ,Research Article ,Signal Transduction ,DNA Replication ,DNA damage ,Morpholines ,Bioengineering ,Library Screening ,Biology ,03 medical and health sciences ,Biomarkers, Tumor ,Genetics ,Humans ,HeLa cells ,Non-coding RNA ,Molecular Biology Techniques ,Protein Kinase Inhibitors ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Molecular Biology Assays and Analysis Techniques ,DNA replication ,Biology and Life Sciences ,DNA ,Cell Biology ,Synthetic Genomics ,Cell cultures ,DNA Replication Fork ,Gene regulation ,Research and analysis methods ,Pyrimidines ,Drug Resistance, Neoplasm ,Cancer cell ,RNA ,Gene expression ,CRISPR-Cas Systems ,Drug Screening Assays, Antitumor ,Genetic screen - Abstract
The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance., Author summary Cancer cells rely on the ATR replication stress response pathway to ensure DNA replication and continued cellular proliferation. As such, inhibitors of the ATR kinase activity represent promising new anti-cancer drugs. However, the tumors’ susceptibility to these drugs can be markedly impacted by their genomic profile. To address this, we employed dual CRISPR knockout and activation genome-wide genetic screens to catalog the genetic determinants of the cellular resistance to multiple ATR inhibitors. We identified several mechanisms which control this resistance, including regulation of apoptosis and stabilization of replication fork stability. Our work lays the foundation for personalized deployment of ATR inhibitors in cancer therapy.
- Published
- 2020
33. PARP10 deficiency manifests by severe developmental delay and DNA repair defect
- Author
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Bassam Abu-Libdeh, George Lucian Moldovan, Orly Elpeleg, Maher Shahrour, Claudia M. Nicolae, Motee Ashhab, Simon Edvardson, Adri M. Galvan, and Daniel Constantin
- Subjects
Male ,0301 basic medicine ,Xeroderma pigmentosum ,DNA Repair ,DNA damage ,DNA repair ,Developmental Disabilities ,Cockayne syndrome ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Proto-Oncogene Proteins ,Exome Sequencing ,Genetics ,medicine ,Humans ,Genetics (clinical) ,biology ,Homozygote ,Brain ,DNA repair protein XRCC4 ,medicine.disease ,Magnetic Resonance Imaging ,Molecular biology ,Pedigree ,Proliferating cell nuclear antigen ,030104 developmental biology ,Child, Preschool ,Mutation ,biology.protein ,Cancer research ,DNA mismatch repair ,Poly(ADP-ribose) Polymerases ,DNA Damage ,Nucleotide excision repair - Abstract
DNA repair mechanisms such as nucleotide excision repair (NER) and translesion synthesis (TLS) are dependent on proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory protein. Recently, homozygosity for p.Ser228Ile mutation in the PCNA gene was reported in patients with neurodegeneration and impaired NER. Using exome sequencing, we identified a homozygous deleterious mutation, c.648delAG, in the PARP10 gene, in a patient suffering from severe developmental delay. In agreement, PARP10 protein was absent from the patient cells. We have previously shown that PARP10 is recruited by PCNA to DNA damage sites and is required for DNA damage resistance. The patient cells were significantly more sensitive to hydroxyurea and UV-induced DNA damage than control cells, resulting in increased apoptosis, indicating DNA repair impairment in the patient cells. PARP10 deficiency joins the long list of DNA repair defects associated with neurodegenerative disorders, including ataxia telangiectasia, xeroderma pigmentosum, Cockayne syndrome, and the recently reported PCNA mutation.
- Published
- 2016
34. Extracellular matrix protein Matrilin-4 regulates stress-induced HSC proliferation via CXCR4
- Author
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Attila Aszodi, Hannah Uckelmann, Sandra Blaszkiewicz, Simon Haas, Marieke A.G. Essers, Claudia M. Nicolae, Raimund Wagener, Stephan Wurzer, and Alexandra Schnell
- Subjects
0301 basic medicine ,Cancer Research ,Receptors, CXCR4 ,Immunology ,Down-Regulation ,Biology ,CXCR4 ,Extracellular matrix ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Stress, Physiological ,Animals ,Matrilin Proteins ,Immunology and Allergy ,Progenitor cell ,Research Articles ,Bone Marrow Transplantation ,Cell Proliferation ,Brief Definitive Report ,Signal transducing adaptor protein ,hemic and immune systems ,Hematopoietic Stem Cells ,Hematopoiesis ,Cell biology ,Mice, Inbred C57BL ,Transplantation ,Haematopoiesis ,030104 developmental biology ,030220 oncology & carcinogenesis ,Female ,Stem cell - Abstract
Essers et al. find that the extracellular matrix adaptor protein Matrilin-4 confers a resistance to stress stimuli in hematopoietic stem cells., During homeostasis, hematopoietic stem cells (HSCs) are mostly kept in quiescence with only minor contribution to steady-state hematopoiesis. However, in stress situations such as infection, chemotherapy, or transplantation, HSCs are forced to proliferate and rapidly regenerate compromised hematopoietic cells. Little is known about the processes regulating this stress-induced proliferation and expansion of HSCs and progenitors. In this study, we identified the extracellular matrix (ECM) adaptor protein Matrilin-4 (Matn4) as an important negative regulator of the HSC stress response. Matn4 is highly expressed in long-term HSCs; however, it is not required for HSC maintenance under homeostasis. In contrast, Matn4 is strongly down-regulated in HSCs in response to proliferative stress, and Matn4 deficiency results in increased proliferation and expansion of HSCs and progenitors after myelosuppressive chemotherapy, inflammatory stress, and transplantation. This enhanced proliferation is mediated by a transient down-regulation of CXCR4 in Matn4−/− HSCs upon stress, allowing for a more efficient expansion of HSCs. Thus, we have uncovered a novel link between the ECM protein Matn4 and cytokine receptor CXCR4 involved in the regulation of HSC proliferation and expansion under acute stress.
- Published
- 2016
35. Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis
- Author
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Claudia M. Nicolae, Attila Aszodi, Paolo Alberton, Raimund Wagener, Maximilian Michael Saller, Juliane Heilig, Zsuzsanna Farkas, Lutz Fleischhauer, Carina Prein, Anja Niehoff, Hauke Clausen-Schaumann, Wolf Christian Prall, and Ping Li
- Subjects
Male ,0301 basic medicine ,Aging ,Osteoarthritis ,Microscopy, Atomic Force ,lcsh:Chemistry ,Extracellular matrix ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,cartilage ,lcsh:QH301-705.5 ,Cells, Cultured ,Spectroscopy ,Mice, Knockout ,General Medicine ,Phenotype ,Computer Science Applications ,Cell biology ,medicine.anatomical_structure ,bone development ,030220 oncology & carcinogenesis ,Knockout mouse ,Female ,Biology ,Article ,Catalysis ,Chondrocyte ,Multiple epiphyseal dysplasia ,Inorganic Chemistry ,03 medical and health sciences ,Chondrocytes ,matrilin ,medicine ,Animals ,Humans ,Matrilin Proteins ,articular cartilage ,Physical and Theoretical Chemistry ,Muscle, Skeletal ,Molecular Biology ,Cell Proliferation ,Cartilage ,Organic Chemistry ,medicine.disease ,Disease Models, Animal ,osteoarthritis ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Proteoglycan ,biology.protein - Abstract
Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4&minus, /&minus, mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4&minus, mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4&minus, mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4&minus, mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.
- Published
- 2020
36. STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability
- Author
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Stephen Mallon, Diana Rose E. Ranoa, Claudia M. Nicolae, Nikolai N. Khodarev, Stephen J. Kron, George Lucian Moldovan, Michael J. Bolt, Xiaona Huang, Ryan C. Widau, A. Parekh, Renate Parry, Ralph R. Weichselbaum, and Ainhoa Arina
- Subjects
0301 basic medicine ,Cancer Research ,Cellular homeostasis ,Biology ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Neoplasms ,Chromosomal Instability ,Animals ,Homeostasis ,Humans ,Cell Proliferation ,Cyclin-dependent kinase 1 ,Innate immune system ,Cell growth ,Membrane Proteins ,Cell cycle ,eye diseases ,Immunity, Innate ,Cell biology ,Sting ,030104 developmental biology ,Cell Transformation, Neoplastic ,Oncology ,030220 oncology & carcinogenesis ,Stimulator of interferon genes ,Cancer cell - Abstract
Given the integral role of stimulator of interferon genes (STING, TMEM173) in the innate immune response, its loss or impairment in cancer is thought to primarily affect antitumor immunity. Here we demonstrate a role for STING in the maintenance of cellular homeostasis through regulation of the cell cycle. Depletion of STING in human and murine cancer cells and tumors resulted in increased proliferation compared with wild-type controls. Microarray analysis revealed genes involved in cell-cycle regulation are differentially expressed in STINGko compared with WT MEFs. STING-mediated regulation of the cell cycle converged on NFκB- and p53-driven activation of p21. The absence of STING led to premature activation of cyclin-dependent kinase 1 (CDK1), early onset to S-phase and mitosis, and increased chromosome instability, which was enhanced by ionizing radiation. These results suggest a pivotal role for STING in maintaining cellular homeostasis and response to genotoxic stress. Significance: These findings provide clear mechanistic understanding of the role of STING in cell-cycle regulation, which may be exploited in cancer therapy because most normal cells express STING, while many tumor cells do not. See related commentary by Gius and Zhu, p. 1295
- Published
- 2018
37. PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress
- Author
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George Lucian Moldovan, Adri M. Galvan, Claudia M. Nicolae, Emily M. Schleicher, and Yuka Imamura-Kawasawa
- Subjects
0301 basic medicine ,DNA Replication ,DNA repair ,Carcinogenesis ,Mice, Nude ,Retinal Pigment Epithelium ,Biology ,Genome Integrity, Repair and Replication ,medicine.disease_cause ,03 medical and health sciences ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,Proto-Oncogene Proteins ,Genetics ,medicine ,Animals ,Humans ,Cell Line, Transformed ,Cell growth ,DNA replication ,DNA Replication Fork ,3. Good health ,Proliferating cell nuclear antigen ,Cell biology ,Neoplasm Proteins ,Up-Regulation ,Replication fork arrest ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer cell ,biology.protein ,Heterografts ,Female ,CRISPR-Cas Systems ,Poly(ADP-ribose) Polymerases ,Cell Division ,DNA Damage ,HeLa Cells - Abstract
During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation, and its overexpression alleviates cellular sensitivity to replication stress and fosters the restart of stalled replication forks. Importantly, mouse xenograft studies showed that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation, potentially by alleviating replication stress and suggest that targeting PARP10 may represent a novel therapeutic approach.
- Published
- 2018
38. PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress
- Author
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Adri M. Galvan, Claudia M. Nicolae, George Lucian Moldovan, and Emily M. Schleicher
- Subjects
biology ,DNA polymerase ,DNA repair ,medicine.disease_cause ,Proliferating cell nuclear antigen ,Cell biology ,Replication fork arrest ,chemistry.chemical_compound ,chemistry ,Cancer cell ,medicine ,biology.protein ,Carcinogenesis ,Gene knockout ,DNA - Abstract
During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and other difficult to replicate regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein PCNA and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation and replication fork elongation. Mechanistically, PARP10 overexpression alleviated cellular sensitivity to replication stress by fostering the restart of stalled replication forks. Importantly, mouse xenograft studies indicated that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation by alleviating replication stress, and suggest that targeting PARP10 may represent a novel therapeutic approach.
- Published
- 2018
39. Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells
- Author
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Hong Gang Wang, Tanay Thakar, Claudia M. Nicolae, Kristen E. Clements, George Lucian Moldovan, and Xinwen Liang
- Subjects
0301 basic medicine ,endocrine system diseases ,Somatic cell ,medicine.medical_treatment ,Genes, BRCA2 ,RAD51 ,Genome Integrity, Repair and Replication ,Piperazines ,Germline ,Gene Knockout Techniques ,0302 clinical medicine ,E2F7 Transcription Factor ,Enzyme Inhibitors ,skin and connective tissue diseases ,0303 health sciences ,Chemistry ,DNA, Neoplasm ,female genital diseases and pregnancy complications ,Neoplasm Proteins ,3. Good health ,030220 oncology & carcinogenesis ,PARP inhibitor ,Poly(ADP-ribose) Polymerases ,medicine.drug ,DNA Replication ,DNA repair ,Poly ADP ribose polymerase ,Antineoplastic Agents ,Biology ,03 medical and health sciences ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,030304 developmental biology ,Cisplatin ,BRCA2 Protein ,Chemotherapy ,Recombinational DNA Repair ,Cancer ,medicine.disease ,Biomarker ,030104 developmental biology ,Cell culture ,Drug Resistance, Neoplasm ,Cancer research ,Phthalazines ,Rad51 Recombinase ,CRISPR-Cas Systems ,Homologous recombination - Abstract
BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.
- Published
- 2018
40. Vegfa regulates perichondrial vascularity and osteoblast differentiation in bone development
- Author
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Claudia M. Nicolae, Yanqiu Liu, Bjorn R. Olsen, Xuchen Duan, Agnes D. Berendsen, and Yurie Murata
- Subjects
Vascular Endothelial Growth Factor A ,Research Report ,endocrine system ,medicine.medical_specialty ,Indian hedgehog ,Cellular differentiation ,Kruppel-Like Transcription Factors ,Neovascularization, Physiologic ,Cell Count ,Biology ,Zinc Finger Protein GLI1 ,Bone and Bones ,Mice ,Calcification, Physiologic ,Osteogenesis ,Internal medicine ,medicine ,Animals ,Perichondrium ,Cell Lineage ,Hedgehog Proteins ,Receptor, Notch2 ,Molecular Biology ,beta Catenin ,Bone Development ,Osteoblasts ,Ossification ,Stem Cells ,Cartilage ,Cell Differentiation ,Osteoblast ,Kinase insert domain receptor ,biology.organism_classification ,Vascular Endothelial Growth Factor Receptor-2 ,Cell biology ,Vascular endothelial growth factor A ,medicine.anatomical_structure ,Endocrinology ,medicine.symptom ,Signal Transduction ,Developmental Biology - Abstract
Vascular endothelial growth factor A (Vegfa) has important roles in endochondral bone formation. Osteoblast precursors, endothelial cells and osteoclasts migrate from perichondrium into primary ossification centers of cartilage templates of future bones in response to Vegfa secreted by (pre)hypertrophic chondrocytes. Perichondrial osteolineage cells also produce Vegfa, but its function is not well understood. By deleting Vegfa in osteolineage cells in vivo, we demonstrate that progenitor-derived Vegfa is required for blood vessel recruitment in perichondrium and the differentiation of osteoblast precursors in mice. Conditional deletion of Vegfa receptors indicates that Vegfa-dependent effects on osteoblast differentiation are mediated by Vegf receptor 2 (Vegfr2). In addition, Vegfa/Vegfr2 signaling stimulates the expression and activity of Indian hedgehog, increases the expression of β-catenin and inhibits Notch2. Our findings identify Vegfa as a regulator of perichondrial vascularity and osteoblast differentiation at early stages of bone development.
- Published
- 2015
41. NFκB regulates p21 expression and controls DNA damage-induced leukemic differentiation
- Author
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George Lucian Moldovan, Claudia M. Nicolae, Daniel Constantin, and Michael J. O’Connor
- Subjects
0301 basic medicine ,Cyclin-Dependent Kinase Inhibitor p21 ,Cancer Research ,Myeloid ,DNA damage ,HL-60 Cells ,Biology ,Cell fate determination ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,medicine ,CRISPR ,Humans ,Binding site ,Promoter Regions, Genetic ,Molecular Biology ,Regulation of gene expression ,Gene Editing ,Binding Sites ,Transcription Factor RelA ,Myeloid leukemia ,NF-kappa B p50 Subunit ,Cell Differentiation ,U937 Cells ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Leukemia, Myeloid ,030220 oncology & carcinogenesis ,CRISPR-Cas Systems ,Tumor Suppressor Protein p53 ,DNA ,DNA Damage - Abstract
DNA damage exposure is a major modifier of cell fate in both normal and cancer tissues. In response to DNA damage, myeloid leukemia cells activate a poorly understood terminal differentiation process. Here, we show that the NFκB pathway directly activates expression of the proliferation inhibitor p21 in response to DNA damage in myeloid leukemia cells. In order to understand the role of this unexpected regulatory event, we ablated the NFκB binding site we identified in the p21 promoter, using CRISPR/Cas9-mediated genome editing. We found that NFκB-mediated p21 activation controls DNA damage-induced myeloid differentiation. Our results uncover a p53-independent pathway for p21 activation involved in controlling hematopoietic cell fate.
- Published
- 2017
42. Heterozygous De Novo UBTF Gain-of-Function Variant Is Associated with Neurodegeneration in Childhood
- Author
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Avraham Shaag, Berivan Baskin, Zöe Powis, Amber Begtrup, Katelyn Payne, Claudia M. Nicolae, Orly Elpeleg, Chitra Prasad, Asuri N. Prasad, Boris Keren, George Lucian Moldovan, Laurie S. Sadler, Caroline Nava, Cyril Mignot, Simon Edvardson, Thomas E. Mullen, and Pankaj B. Agrawal
- Subjects
0301 basic medicine ,Adult ,Male ,Adolescent ,Nucleolus ,18S ,Biology ,Ribosome ,Polymorphism, Single Nucleotide ,Pediatrics ,Promoter Regions ,03 medical and health sciences ,Young Adult ,Genetic ,Report ,Genetics ,RNA polymerase I ,RNA, Ribosomal, 18S ,Humans ,Polymorphism ,Child ,Promoter Regions, Genetic ,Transcription factor ,Genetics (clinical) ,Ribosomal ,Brain Diseases ,Brain ,Neurodegenerative Diseases ,Single Nucleotide ,Ribosomal RNA ,Molecular biology ,Chromatin ,External transcribed spacer ,DNA-Binding Proteins ,030104 developmental biology ,Transcription preinitiation complex ,RNA ,Female ,Atrophy ,Pol1 Transcription Initiation Complex Proteins ,Cell Nucleolus - Abstract
Summary Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5–7 years, we identified a heterozygous variant, c.628G>A in UBTF , encoding p.Glu210Lys in UBF, which occurred de novo in all cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 5′- external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism.
- Published
- 2017
43. The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance
- Author
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Georgios I. Karras, Subhajyoti De, Claudia M. Nicolae, Alexander H.S. Vlahos, Katherine N. Choe, George Lucian Moldovan, and Erin R. Aho
- Subjects
DNA Replication ,DNA repair ,Eukaryotic DNA replication ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,DNA polymerase delta ,Genomic Instability ,Replication factor C ,Proliferating Cell Nuclear Antigen ,Proto-Oncogene Proteins ,Humans ,Molecular Biology ,Replication protein A ,DNA clamp ,biology ,Sumoylation ,Cell Biology ,Molecular biology ,Proliferating cell nuclear antigen ,biology.protein ,DNA mismatch repair ,Poly(ADP-ribose) Polymerases ,Protein Processing, Post-Translational ,DNA Damage ,HeLa Cells - Abstract
All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.
- Published
- 2014
44. ERKing Trypanosoma: PCNA phosphorylation as novel target
- Author
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Claudia M. Nicolae and George Lucian Moldovan
- Subjects
Trypanosoma ,biology.protein ,Phosphorylation ,Cell Biology ,Biology ,Trypanosoma brucei ,biology.organism_classification ,Molecular Biology ,Developmental Biology ,Cell biology ,Proliferating cell nuclear antigen - Published
- 2016
45. Growth of cranial synchondroses and sutures requires polycystin-1
- Author
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Naomi Fukai, Claudia M. Nicolae, Bjorn R. Olsen, Brandeis McBratney-Owen, Jing Zhou, Elona Kolpakova-Hart, and Bo Hou
- Subjects
TRPP Cation Channels ,Blotting, Western ,Synchondrosis ,Biology ,Article ,Craniosynostosis ,Mice ,03 medical and health sciences ,0302 clinical medicine ,In Situ Nick-End Labeling ,medicine ,Animals ,Cranial base ,Craniofacial ,Molecular Biology ,In Situ Hybridization ,Polycystin-1 ,030304 developmental biology ,Suture ,Mice, Knockout ,Fibrous joint ,0303 health sciences ,Craniofacial skeleton ,Rostrum ,Gene Expression Regulation, Developmental ,Neural crest ,Cranial Sutures ,Anatomy ,Cell Biology ,medicine.disease ,Embryonic stem cell ,Skull ,medicine.anatomical_structure ,Bromodeoxyuridine ,Neural Crest ,embryonic structures ,030217 neurology & neurosurgery ,Signal Transduction ,Developmental Biology - Abstract
In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies. Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.
- Published
- 2008
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46. Abnormal Collagen Fibrils in Cartilage of Matrilin-1/Matrilin-3-deficient Mice
- Author
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Mats Paulsson, Ernst B. Hunziker, Lukas Enggist, Nicolai Miosge, Daniel Studer, Ya-Ping Ko, Attila Aszódi, Raimund Wagener, Claudia M. Nicolae, and Anja Niehoff
- Subjects
Cartilage, Articular ,Mice, Inbred Strains ,Cartilage Oligomeric Matrix Protein ,Matrix (biology) ,Biochemistry ,Bone and Bones ,Extracellular matrix ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Matrilin Proteins ,Molecular Biology ,Crosses, Genetic ,Glycoproteins ,030304 developmental biology ,Mice, Knockout ,Extracellular Matrix Proteins ,0303 health sciences ,Bone Development ,Chemistry ,Cartilage ,Homozygote ,Signal transducing adaptor protein ,Fibrillogenesis ,Cell Biology ,Anatomy ,Cell biology ,Mice, Inbred C57BL ,Collagen, type I, alpha 1 ,medicine.anatomical_structure ,Animals, Newborn ,Epiphysis ,030220 oncology & carcinogenesis ,Ultrastructure ,Collagen ,Tomography, X-Ray Computed - Abstract
Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.
- Published
- 2007
47. HUWE1 interacts with PCNA to alleviate replication stress
- Author
-
George Lucian Moldovan, Katherine N. Choe, Claudia M. Nicolae, Subhajyoti De, Veronique A. J. Smits, Raimundo Freire, Maria Rocio Delgado‐Diaz, Yuka Imamura Kawasawa, and Daniel Constantin
- Subjects
0301 basic medicine ,DNA Replication ,DNA Repair ,Ubiquitin-Protein Ligases ,Eukaryotic DNA replication ,Biology ,Pre-replication complex ,Biochemistry ,Genomic Instability ,Cell Line ,DNA replication factor CDT1 ,Histones ,03 medical and health sciences ,Gene Knockout Techniques ,Replication factor C ,Control of chromosome duplication ,Minichromosome maintenance ,Stress, Physiological ,Proliferating Cell Nuclear Antigen ,Genetics ,Humans ,News & Views ,Molecular Biology ,Ubiquitin ,Tumor Suppressor Proteins ,DNA replication ,Ubiquitination ,Articles ,Protein Transport ,030104 developmental biology ,Phenotype ,biology.protein ,Origin recognition complex ,Protein Processing, Post-Translational ,DNA Damage ,Protein Binding - Abstract
Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.
- Published
- 2015
48. Loss of α10β1 integrin expression leads to moderate dysfunction of growth plate chondrocytes
- Author
-
Claudia M. Nicolae, Therese Bengtsson, Reinhard Fässler, Ernst B. Hunziker, Attila Aszódi, and Evy Lundgren-Åkerlund
- Subjects
Integrins ,Genotype ,Transgene ,Green Fluorescent Proteins ,Integrin ,Morphogenesis ,Mice, Transgenic ,Bone and Bones ,Chondrocyte ,Collagen receptor ,Mice ,Chondrocytes ,Cell Adhesion ,medicine ,Animals ,Immunoprecipitation ,Growth Plate ,Coloring Agents ,Cell adhesion ,Alleles ,In Situ Hybridization ,Cell Proliferation ,Bone Development ,Models, Genetic ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Cell growth ,Integrin beta1 ,Cartilage ,Cell Biology ,Cell biology ,Mice, Inbred C57BL ,Molecular Weight ,Microscopy, Electron ,medicine.anatomical_structure ,Animals, Newborn ,Immunology ,biology.protein ,RNA ,Collagen ,Integrin alpha Chains ,Gene Deletion - Abstract
Integrin alpha10beta1 is a collagen-binding integrin expressed on chondrocytes. In order to unravel the role of the alpha10 integrin during development, we generated mice carrying a constitutive deletion of the alpha10 integrin gene. The mutant mice had a normal lifespan and were fertile but developed a growth retardation of the long bones. Analysis of the skeleton revealed defects in the growth plate after birth characterized by a disturbed columnar arrangement of chondrocytes, abnormal chondrocyte shape and reduced chondrocyte proliferation. Electron microscopy of growth plates from newborn mice revealed an increased number of apoptotic chondrocytes and reduced density of the collagen fibrillar network compared to these structures in control mice. These results demonstrate that integrin alpha10beta1 plays a specific role in growth plate morphogenesis and function.
- Published
- 2005
49. Erratum to: PARP10 deficiency manifests by severe developmental delay and DNA repair defect
- Author
-
Orly Elpeleg, Adri M. Galvan, Motee Ashhab, Bassam Abu-Libdeh, George Lucian Moldovan, Simon Edvardson, Daniel Constantin, Claudia M. Nicolae, and Maher Shahrour
- Subjects
Genetics ,0301 basic medicine ,business.industry ,DNA repair ,Published Erratum ,MEDLINE ,Neurogenetics ,Computational biology ,01 natural sciences ,Article ,Human genetics ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,030104 developmental biology ,Medicine ,business ,Neuroscience ,Genetics (clinical) - Abstract
DNA repair mechanisms such as Nucleotide Excision Repair (NER) and Translesion Synthesis (TLS) are dependent on proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory protein. Recently, homozygosity for p.Ser228Ile mutation in the PCNA gene was reported in patients with neurodegeneration and impaired NER. Using exome sequencing we identified a homozygous deleterious mutation, c.648delAG, in the PARP10 gene, in a patient suffering from infantile neurodegeneration. In agreement, PARP10 protein was absent from the patient cells. We have previously shown that PARP10 is recruited by PCNA to DNA damage sites and is required for DNA damage resistance. The patient cells were significantly more sensitive to hydroxyurea and UV-induced DNA damage than control cells, resulting in increased apoptosis, indicating DNA repair impairment in the patient cells. PARP10 deficiency joins the long list of DNA repair defects associated with neurodegenerative disorders, including ataxia telangiectasia, xeroderma pigmentosum, Cockayne syndrome and the recently reported PCNA mutation.
- Published
- 2016
50. PARI overexpression promotes genomic instability and pancreatic tumorigenesis
- Author
-
Alan D. D'Andrea, Claudia M. Nicolae, George Lucian Moldovan, Donniphat Dejsuphong, Eunmi Park, Alec C. Kimmelman, and Kevin W O'Connor
- Subjects
Genome instability ,DNA Replication ,Cancer Research ,DNA Repair ,DNA repair ,DNA damage ,Gene Expression ,Biology ,medicine.disease_cause ,Genomic Instability ,Article ,S Phase ,Mice ,Pancreatic cancer ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Homologous Recombination ,Cell Proliferation ,Cell growth ,medicine.disease ,Molecular biology ,Xenograft Model Antitumor Assays ,Homologous Recombination Pathway ,Tumor Burden ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Pancreatic Neoplasms ,Cell Transformation, Neoplastic ,Oncology ,Cancer research ,Homologous recombination ,Carcinogenesis ,Carrier Proteins ,DNA Damage - Abstract
Treatment options for patients with pancreatic ductal adenocarcinoma (PDAC) remain limited. Therapeutic targets of interest include mutated molecules that predispose to pancreatic cancer such as KRAS and TP53. Here, we show that an element of the homologous recombination pathway of DNA repair, the PARP-binding protein C12orf48/PARI (PARPBP), is overexpressed specifically in pancreatic cancer cells where it is an appealing candidate for targeted therapy. PARI upregulation in pancreatic cancer cells or avian DT40 cells conferred DNA repair deficiency and genomic instability. Significantly, PARI silencing compromised cancer cell proliferation in vitro, leading to cell-cycle alterations associated with S-phase delay, perturbed DNA replication, and activation of the DNA damage response pathway in the absence of DNA damage stimuli. Conversely, PARI overexpression produced tolerance to DNA damage by promoting replication of damaged DNA. In a mouse xenograft model of pancreatic cancer, PARI silencing was sufficient to reduce pancreatic tumor growth in vivo. Taken together, our findings offered a preclinical proof-of-concept for PARI as candidate therapeutic target to treat PDAC. Cancer Res; 73(8); 2529–39. ©2013 AACR.
- Published
- 2013
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