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PPM1D suppresses p53-dependent transactivation and cell death by inhibiting the HRI-eIF2alpha-ATF4 pathway [RNA-seq]

ABSTRACT: The p53 transcription factor is a master regulator of cellular stress responses restrained by repressors such as MDM2 and the phosphatase PPM1D. Activation of p53 with pharmacological inhibitors of its repressors is being tested in clinical trials for cancer therapy, but efficacy has been limited by poor induction of tumor cell death. We demonstrate that dual inhibition of MDM2 and PPM1D induces cell death in multiple cancer cell types via amplification of the p53 transcriptional program through the eIF2alpha -ATF4 pathway. PPM1D inhibition induces phosphorylation of eIF2alpha, ATF4 accumulation, and ATF4-dependent enhancement of p53-dependent transactivation upon MDM2 inhibition. Dual inhibition of p53 repressors depletes heme and induces HRI-dependent eIF2alpha phosphorylation. Pharmacological induction of eIF2alpha phosphorylation synergizes with MDM2 inhibition to induce cell death and halt tumor growth in mice. These results demonstrate that PPM1D inhibits both the p53 network and the integrated stress response controlled by eIF2alpha, with clear therapeutic implications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE191148 | GEO | 2022/11/01

REPOSITORIES: GEO

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PPM1D suppresses p53-dependent transactivation and cell death by inhibiting the HRI-eIF2alpha-ATF4 pathway [ChIP-seq]

Project description:The p53 transcription factor is a master regulator of cellular stress responses restrained by repressors such as MDM2 and the phosphatase PPM1D. Activation of p53 with pharmacological inhibitors of its repressors is being tested in clinical trials for cancer therapy, but efficacy has been limited by poor induction of tumor cell death. We demonstrate that dual inhibition of MDM2 and PPM1D induces cell death in multiple cancer cell types via amplification of the p53 transcriptional program through the eIF2alpha -ATF4 pathway. PPM1D inhibition induces phosphorylation of eIF2alpha, ATF4 accumulation, and ATF4-dependent enhancement of p53-dependent transactivation upon MDM2 inhibition. Dual inhibition of p53 repressors depletes heme and induces HRI-dependent eIF2alpha phosphorylation. Pharmacological induction of eIF2alpha phosphorylation synergizes with MDM2 inhibition to induce cell death and halt tumor growth in mice. These results demonstrate that PPM1D inhibits both the p53 network and the integrated stress response controlled by eIF2alpha, with clear therapeutic implications.

2022-09-01 | GSE191149 | GEO

Cooperation of Nutlin-3a and a Wip1 inhibitor to induce p53 activity

Project description:Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists.

2016-05-02 | GSE80716 | GEO

PPM1D mutations are oncogenic drivers of Diffuse Midline Gliomas and are sufficient to enhance glioma formation

Project description:The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genomes sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of DMGs and are enriched in primary pontine tumors. Through the development of novel DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D activity is required for in vivo oncogenesis. Finally, we applied integrative phosphoproteomic and functional genomics assays and found that the oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition. Together, these findings highlight PPM1D mutations to represent a targetable driver of pediatric gliomas.

2021-07-11 | GSE179813 | GEO

Pharmacological inhibition of WIP1 sensitizes acute myeloid leukemia cells to MDM2 inhibitor Nutlin-3a

Project description:The wild-type p53-inducible protein phosphatase WIP1, encoded by the PPM1D gene, is a negative regulator of p53 and is involved in cell cycle control and DNA damage stress-response. In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in a number of combination strategies. We investigated the therapeutic potential of its inhibition through the selective WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in a panel of AML cell lines and performed gene expression analysis of single agent and combined treatments. A p53-related signature was significantly upregulated in the TP53-wildtype MV-4-11 AML cell line. Moreover, drug-specific transcriptional changes likely contribute to the synergistic combination effect.

2021-05-06 | GSE156182 | GEO

Gene expression profiling of HepG2 cell response to bortezomib in combination with nelfinavir or ISRIB, compounds targeting the eIF2a-ATF4 pathway

Project description:We investigate how modulation of the eIF2alpha-ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the highest upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2alpha-ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP-ATF-driven transcription, augments ER stress and cell death in cells exposed to bortezomib, while TRIB3 overexpression enhances the cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2alpha-ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib.

2021-05-24 | GSE166923 | GEO

Substrate spectrum of PPM1D in the cellular response to DNA double strand breaks

Project description:PPM1D (also known as WIP1) is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) by dephosphorylation of DDR proteins. Amplification of PPM1D gene or gain-of-function mutations are commonly found in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double strand breaks (DSBs) by topoisomerase II poison etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription and RNA processing. One third of DSB-induced S/TQ phosphorylation sites are dephosphorylated by PPM1D, demonstrating that PPM1D only partially counteracts ATM/ATR/DNA-PK signaling. PPM1D-targeted phosphorylation sites are found in a specific amino acid sequence motif that is characterized by glutamic acid residues, high intrinsic disorder and poor evolutionary conservation. We identified a functionally uncharacterized protein Kanadaptin as ATM and PPM1D substrate upon DSB induction. We propose that PPM1D plays a role during the response to DSBs formation by regulating the phosphorylation of DNA- and RNA-binding proteins in intrinsically disordered regions.

2022-09-06 | PXD035420 | Pride

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA repair at damaged telomeres

Project description:Protein phosphatase magnesium-dependent 1 delta (PPM1D) is involved in termination of the cell cycle checkpoint by counteracting activity of the tumour suppressor protein p53. By dephosphorylating various proteins at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified interaction between PPM1D and the components of the shelterin complex that protects telomeric DNA. Interaction between PPM1D and TRF2 was confirmed by immunoprecipitation and proximity ligation assay. Confocal microscopy revealed colocalisation of the endogenous PPM1D with TRF2 at telomeres. Further, we found that TRF2 was phosphorylated by ATR at S410 after induction of DNA damage at telomeres and this modification was increased in cells lacking PPM1D or after PPM1D inhibition. Phosphorylation of TRF2 stimulated its interaction with TIN2 at telomeres and in vitro. Conversely, overexpression of PPM1D impaired localisation of TIN2 at telomeres. Finally, inhibition of PPM1D impaired recruitment of 53BP1 and RAD51 to DNA double strand breaks at telomeres. Expression of TRF2-S410A mutant fully rescued the recruitment of 53BP1 to telomeric breaks. These results suggest that TRF2 phosphorylation promotes association of TIN2 with the shelterin complex and regulates DNA repair at telomeres.

2023-03-11 | PXD035268 | Pride

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA repair at damaged telomeres.

Project description: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 colocalized with TRF2 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.

2023-03-11 | PXD035273 | Pride

Targeting MDM2-induced p53 upregulation to enhance anti-leukemia immunity post allogeneic stem cell transplantation

Project description:Relapse after allo-HCT is a major cause of death of AML patients and results from immune evasion of AML blasts. Dysfunction of the p53 signaling pathway is frequent in AML and often caused by upregulation of the central p53 negative regulator Murine Double Minute 2 (MDM2). Besides its oncogenic effects p53 also regulates immune function and immune surveillance of solid cancer. We hypothesize that p53 also controls immune-related genes in AML cells and that p53 reactivation via MDM2-inhibition may enhance the immunogenicity of AML cells to allogeneic T cells.

2022-09-20 | GSE158103 | GEO

Specific translation during aging mediated by phosphorylation of Schizosaccharomyces pombe eIF2alpha

Project description:Aging is a highly regulated process in eukaryotes. Many biochemical pathways are affected by aging and, among them, protein homeostasis is one key process. Our hypothesis is that protein translation may be important to maintain a correct balance of active, properly folded proteins in the cell and, therefore, help supporting a normal longevity. Phosphorylation of eIF2alpha is one of the most important regulatory steps in protein translation in eukaryotes, reducing total protein translation and promoting the specific production of some proteins (Gcn4, Atf4, etc) We decided to test whether eIF2alpha phosphorylation has specific roles in protein translation during aging in Schizosaccharomyces pombe

2021-04-19 | GSE155256 | GEO

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