In response to DNA replication stress, DNA damage signaling kinases inhibit origin firing and promote the remodeling and stabilization of replication forks, leading to a systemic reduction in DNA synthesis that protects genomic integrity. Little is understood about the regulatory mechanisms of replication stress recovery, including the mechanisms involved in the restart of stalled replication forks. Here, we identify the oncogenic phosphatase PPM1D/WIP1 as a critical regulator of replication fork restart. Upon recovery from replication stress, PPM1D prevents excessive MRE11- and DNA2-dependent nucleolytic degradation of stalled forks. Loss of PPM1D function leads to defects in RAD51 recruitment to chromatin and impairs RAD51-dependent fork restart. Phosphoproteomic analysis reveals that PPM1D regulates a network of ATM substrates, several of which are phosphorylated at an S/T-Q-(E/D)n motif. Strikingly, inhibition of ATM suppresses the deleterious consequences of impaired PPM1D function at replication forks, enabling timely fork restart. The dominant effect of ATM hyper-signaling in suppressing fork restart occurs, in part, through the excessive engagement of 53BP1 and consequent RAD51 antagonization. These findings uncover a new mode of ATM signaling responding to fork stalling and highlights the need for PPM1D to restrain ATM signaling and enable proper fork restart.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

• MeSH
• buněčná smrt genetika   MeSH
• lidé MeSH
• myši MeSH
• nádorová transformace buněk * genetika   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• poškození DNA * genetika   MeSH
• proliferace buněk genetika   MeSH
• proteinfosfatasa 2C * genetika   metabolismus   MeSH
• proteinfosfatasy genetika   metabolismus   MeSH
• stárnutí buněk * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH
• PPM1D protein, human MeSH Prohlížeč
• proteinfosfatasa 2C * MeSH
• proteinfosfatasy MeSH

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

• Klíčová slova
• PPM1D phosphatase, cancer, cell cycle, cyclin E1, replication stress,
• MeSH
• cyklin E genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * genetika   metabolismus   MeSH
• nádory * MeSH
• nestabilita genomu MeSH
• proteinfosfatasa 2C genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cyklin E MeSH
• nádorový supresorový protein p53 * MeSH
• PPM1D protein, human MeSH Prohlížeč
• proteinfosfatasa 2C MeSH

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

• MeSH
• fosforylace MeSH
• lidé MeSH
• poškození DNA MeSH
• protein TRF2 * MeSH
• proteiny vázající telomery * metabolismus   MeSH
• proteomika MeSH
• shelterinový komplex * MeSH
• telomery metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein TRF2 * MeSH
• proteiny vázající telomery * MeSH
• shelterinový komplex * MeSH
• TINF2 protein, human MeSH Prohlížeč

Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/- mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.

• Klíčová slova
• cancer, cell-cycle checkpoint, protein phosphatase, tumor suppressor p53,
• MeSH
• apoptóza * MeSH
• buněčný cyklus MeSH
• ionizující záření MeSH
• lymfom metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory vyvolané zářením metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• proliferace buněk MeSH
• proteinfosfatasa 2C fyziologie   MeSH
• thymocyty cytologie   metabolismus   MeSH
• thymus * cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH
• Ppm1d protein, mouse MeSH Prohlížeč
• proteinfosfatasa 2C MeSH
• Trp53 protein, mouse MeSH Prohlížeč

The most common histological subtypes of cutaneous melanoma include superficial spreading and nodular melanoma. However, the spectrum of somatic mutations developed in those lesions and all potential druggable targets have not yet been fully elucidated. We present the results of a sequence capture NGS analysis of 114 primary nodular and superficial spreading melanomas identifying driver mutations using biostatistical, immunohistochemical and/or functional approach. The spectrum and frequency of pathogenic or likely pathogenic variants were identified across 54 evaluated genes, including 59 novel mutations, and the newly identified TP53 loss-of-function mutations p.(L194P) and p.(R280K). Frequently mutated genes most commonly affected the MAPK pathway, followed by chromatin remodeling, and cell cycle regulation. Frequent aberrations were also detected in the genes coding for proteins involved in DNA repair and the regulation and modification of cellular tight junctions. Furthermore, relatively frequent mutations were described in KDR and MET, which represent potential clinically important targets. Those results suggest that with the development of new therapeutic possibilities, not only BRAF testing, but complex molecular testing of cutaneous melanoma may become an integral part of the decision process concerning the treatment of patients with melanoma.

• MeSH
• buněčný cyklus genetika   MeSH
• dospělí MeSH
• frekvence genu genetika   MeSH
• genetická predispozice k nemoci genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• maligní melanom kůže MeSH
• MAP kinasový signální systém genetika   MeSH
• melanom genetika   patologie   MeSH
• mladý dospělý MeSH
• mutace ztráty funkce genetika   MeSH
• nádorové biomarkery genetika   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory kůže genetika   patologie   MeSH
• oprava DNA genetika   MeSH
• protoonkogenní proteiny B-Raf genetika   MeSH
• restrukturace chromatinu genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• těsný spoj genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové biomarkery MeSH
• nádorový supresorový protein p53 MeSH
• protoonkogenní proteiny B-Raf MeSH
• TP53 protein, human MeSH Prohlížeč

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1DT allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1DT resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apcmin mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcminPpm1dT/+ mice were less sensitive to 5-fluorouracil when compared to ApcminPpm1d+/+and the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.

• MeSH
• chromatin účinky léků   MeSH
• exony genetika   MeSH
• fluoruracil farmakologie   MeSH
• karcinogeneze účinky léků   MeSH
• kontrolní body buněčného cyklu genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• myši MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory tračníku farmakoterapie   genetika   patologie   MeSH
• oprava DNA účinky léků   MeSH
• poškození DNA účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• protein familiární adenomatózní polypózy genetika   MeSH
• proteinfosfatasa 2C genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenomatous polyposis coli protein, mouse MeSH Prohlížeč
• chromatin MeSH
• fluoruracil MeSH
• nádorový supresorový protein p53 MeSH
• PPM1D protein, human MeSH Prohlížeč
• protein familiární adenomatózní polypózy MeSH
• proteinfosfatasa 2C MeSH
• Trp53 protein, mouse MeSH Prohlížeč

Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.

• Klíčová slova
• DNA repair, PARP inhibitor, chemotherapy, genotoxic stress, olaparib, phosphatase,
• MeSH
• 53BP1 metabolismus   MeSH
• apoptóza účinky léků   MeSH
• chemorezistence účinky léků   MeSH
• chromatin metabolismus   MeSH
• ftalaziny farmakologie   MeSH
• HEK293 buňky MeSH
• homologní rekombinace genetika   MeSH
• kontrolní body fáze G2 buněčného cyklu MeSH
• kontrolní body fáze S buněčného cyklu MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu metabolismus   MeSH
• oprava DNA genetika   fyziologie   MeSH
• PARP inhibitory farmakologie   MeSH
• piperaziny farmakologie   MeSH
• poškození DNA genetika   fyziologie   MeSH
• proliferace buněk účinky léků   MeSH
• protein BRCA1 metabolismus   MeSH
• proteinfosfatasa 2C antagonisté a inhibitory   genetika   metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• BRCA1 protein, human MeSH Prohlížeč
• chromatin MeSH
• ftalaziny MeSH
• olaparib MeSH Prohlížeč
• PARP inhibitory MeSH
• piperaziny MeSH
• PPM1D protein, human MeSH Prohlížeč
• protein BRCA1 MeSH
• proteinfosfatasa 2C MeSH
• protinádorové látky MeSH
• TP53BP1 protein, human MeSH Prohlížeč