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• W3016004654 abstract "•Overexpressed PGAM1 in gliomas sequesters the WIP1 phosphatase in the cytoplasm•PGAM1 prevents nuclear translocation and deactivation of DNA damage repair by WIP1•Blocking PGAM1:WIP1 binding restores WIP1 localization to deactivate ATM signaling•Silencing of PGAM1 improves therapeutic outcome of IR and TMZ in gliomas The metabolic enzyme phosphoglycerate mutase 1 (PGAM1) is overexpressed in several types of cancer, suggesting an additional function beyond its established role in the glycolytic pathway. We here report that PGAM1 is overexpressed in gliomas where it increases the efficiency of the DNA damage response (DDR) pathway by cytoplasmic binding of WIP1 phosphatase, thereby preventing WIP1 nuclear translocation and subsequent dephosphorylation of the ATM signaling pathway. Silencing of PGAM1 expression in glioma cells consequently decreases formation of γ-H2AX foci, increases apoptosis, and decreases clonogenicity following irradiation (IR) and temozolomide (TMZ) treatment. Furthermore, mice intracranially implanted with PGAM1-knockdown cells have significantly improved survival after treatment with IR and TMZ. These effects are counteracted by exogenous expression of two kinase-dead PGAM1 mutants, H186R and Y92F, indicating an important non-enzymatic function of PGAM1. Our findings identify PGAM1 as a potential therapeutic target in gliomas. The metabolic enzyme phosphoglycerate mutase 1 (PGAM1) is overexpressed in several types of cancer, suggesting an additional function beyond its established role in the glycolytic pathway. We here report that PGAM1 is overexpressed in gliomas where it increases the efficiency of the DNA damage response (DDR) pathway by cytoplasmic binding of WIP1 phosphatase, thereby preventing WIP1 nuclear translocation and subsequent dephosphorylation of the ATM signaling pathway. Silencing of PGAM1 expression in glioma cells consequently decreases formation of γ-H2AX foci, increases apoptosis, and decreases clonogenicity following irradiation (IR) and temozolomide (TMZ) treatment. Furthermore, mice intracranially implanted with PGAM1-knockdown cells have significantly improved survival after treatment with IR and TMZ. These effects are counteracted by exogenous expression of two kinase-dead PGAM1 mutants, H186R and Y92F, indicating an important non-enzymatic function of PGAM1. Our findings identify PGAM1 as a potential therapeutic target in gliomas. Transformation of normal cells into rapidly proliferating cancer cells requires alterations of cell cycle checkpoints that act to regulate cellular proliferation (Hartwell and Kastan, 1994Hartwell L.H. Kastan M.B. Cell cycle control and cancer.Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2309) Google Scholar). DNA damage repair (DDR) is one of the major pathways that prevent normal cells from transforming into cancer cells. In normal cells, the DDR pathway is activated upon exposure to agents that induce DNA damage that require repair. The DDR pathway is deactivated once the DNA repair process is complete (Kim and Haber, 2009Kim J.A. Haber J.E. Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete.Proc. Natl. Acad. Sci. U S A. 2009; 106: 1151-1156Crossref PubMed Scopus (80) Google Scholar); if the DNA lesion cannot be repaired, the cell undergoes cellular senescence or apoptosis. In cancer cells, however, the pathway is constitutively active. Current cancer treatments, such as irradiation (IR) and chemotherapy, attempt to induce DNA lesions to levels that surpass the ability of the cell to repair all of the DNA damage, ultimately leading to cell death (Curtin, 2013Curtin N.J. Inhibiting the DNA damage response as a therapeutic manoeuvre in cancer.Br. J. Pharmacol. 2013; 169: 1745-1765Crossref PubMed Scopus (59) Google Scholar). Cancer cells, however, are often efficient in repairing the DNA damage and thus can avoid senescence and cell death. Recent findings suggest that cellular metabolism is altered in cancer cells in order to support the needs of indefinite proliferation by performing functions that are not fully understood (DeBerardinis and Chandel, 2016DeBerardinis R.J. Chandel N.S. Fundamentals of cancer metabolism.Sci. Adv. 2016; 2: e1600200Crossref PubMed Scopus (1482) Google Scholar, Pavlova and Thompson, 2016Pavlova N.N. Thompson C.B. The emerging hallmarks of cancer metabolism.Cell Metab. 2016; 23: 27-47Abstract Full Text Full Text PDF PubMed Scopus (2918) Google Scholar). Several enzymes in the glycolytic pathway, such as hexose kinase II (HKII), pyruvate kinase-M2 (PKM2), phospho-fructo kinase (PFK), enolase, and phosphoglycerate mutase (PGAM1), are aberrantly overexpressed in cancer cells (Wolf et al., 2011Wolf A. Agnihotri S. Micallef J. Mukherjee J. Sabha N. Cairns R. Hawkins C. Guha A. Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme.J. Exp. Med. 2011; 208: 313-326Crossref PubMed Scopus (547) Google Scholar, Mukherjee et al., 2013Mukherjee J. Phillips J.J. Zheng S. Wiencke J. Ronen S.M. Pieper R.O. Pyruvate kinase M2 expression, but not pyruvate kinase activity, is up-regulated in a grade-specific manner in human glioma.PLoS ONE. 2013; 8: e57610Crossref PubMed Scopus (71) Google Scholar, Mukherjee et al., 2016Mukherjee J. Ohba S. See W.L. Phillips J.J. Molinaro A.M. Pieper R.O. PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells.Int. J. Cancer. 2016; 139: 99-111Crossref PubMed Scopus (25) Google Scholar, Capello et al., 2011Capello M. Ferri-Borgogno S. Cappello P. Novelli F. α-Enolase: a promising therapeutic and diagnostic tumor target.FEBS J. 2011; 278: 1064-1074Crossref PubMed Scopus (192) Google Scholar, Vander Heiden et al., 2010Vander Heiden M.G. Locasale J.W. Swanson K.D. Sharfi H. Heffron G.J. Amador-Noguez D. Christofk H.R. Wagner G. Rabinowitz J.D. Asara J.M. Cantley L.C. Evidence for an alternative glycolytic pathway in rapidly proliferating cells.Science. 2010; 329: 1492-1499Crossref PubMed Scopus (499) Google Scholar). PGAM1 converts 3-phosphoglycerate (3-PG) into 2-phosphoglycerate (2-PG) using phospho-histidine11 as a phosphate donor/acceptor site within its catalytic domain through formation of a 2,3-bisphosphoglycerate intermediate (Vander Heiden et al., 2010Vander Heiden M.G. Locasale J.W. Swanson K.D. Sharfi H. Heffron G.J. Amador-Noguez D. Christofk H.R. Wagner G. Rabinowitz J.D. Asara J.M. Cantley L.C. Evidence for an alternative glycolytic pathway in rapidly proliferating cells.Science. 2010; 329: 1492-1499Crossref PubMed Scopus (499) Google Scholar, Hitosugi et al., 2012Hitosugi T. Zhou L. Elf S. Fan J. Kang H.B. Seo J.H. Shan C. Dai Q. Zhang L. Xie J. et al.Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.Cancer Cell. 2012; 22: 585-600Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). PGAM1 is expressed at various levels within various normal tissues during cellular differentiation or transformation (Jiang et al., 2014Jiang X. Sun Q. Li H. Li K. Ren X. The role of phosphoglycerate mutase 1 in tumor aerobic glycolysis and its potential therapeutic implications.Int. J. Cancer. 2014; 135: 1991-1996Crossref PubMed Scopus (47) Google Scholar). Furthermore, PGAM1 is overexpressed in several types of cancer, including gliomas (Sanzey et al., 2015Sanzey M. Abdul Rahim S.A. Oudin A. Dirkse A. Kaoma T. Vallar L. Herold-Mende C. Bjerkvig R. Golebiewska A. Niclou S.P. Comprehensive analysis of glycolytic enzymes as therapeutic targets in the treatment of glioblastoma.PLoS ONE. 2015; 10: e0123544Crossref PubMed Scopus (75) Google Scholar, Liu et al., 2018Liu Z.G. Ding J. Du C. Xu N. Wang E.L. Li J.Y. Wang Y.Y. Yu J.M. Phosphoglycerate mutase 1 is highly expressed in C6 glioma cells and human astrocytoma.Oncol. Lett. 2018; 15: 8935-8940PubMed Google Scholar). PGAM1 is unique among the glycolytic enzymes in that its transcription is regulated by the tumor suppressor p53 (Cheung and Vousden, 2010Cheung E.C. Vousden K.H. The role of p53 in glucose metabolism.Curr. Opin. Cell Biol. 2010; 22: 186-191Crossref PubMed Scopus (73) Google Scholar), and increased expression of PGAM1 has been reported to immortalize primary cells through an unknown mechanism (Kondoh et al., 2005Kondoh H. Lleonart M.E. Gil J. Wang J. Degan P. Peters G. Martinez D. Carnero A. Beach D. Glycolytic enzymes can modulate cellular life span.Cancer Res. 2005; 65: 177-185Crossref PubMed Google Scholar). Cancer cells that express PKM2 have increased levels of phosphorylated PGAM1 at residue histidine11, which leads to increased mutase activity and results in increased production of PEP. This positive feedback loop of PEP production and enzymatic activity of PGAM1 may act as an alternate route to funnel more metabolites into the biosynthetic arm of the glycolysis pathway (Vander Heiden et al., 2010Vander Heiden M.G. Locasale J.W. Swanson K.D. Sharfi H. Heffron G.J. Amador-Noguez D. Christofk H.R. Wagner G. Rabinowitz J.D. Asara J.M. Cantley L.C. Evidence for an alternative glycolytic pathway in rapidly proliferating cells.Science. 2010; 329: 1492-1499Crossref PubMed Scopus (499) Google Scholar). The benefit of PGAM1 overexpression was attributed toward increased glycolysis and biosynthesis via the pentose phosphate pathway (PPP), thereby promoting cancer cell proliferation and tumor growth that can be reversed using genetic and pharmacological approaches to inhibit PGAM1 activity (Hitosugi et al., 2012Hitosugi T. Zhou L. Elf S. Fan J. Kang H.B. Seo J.H. Shan C. Dai Q. Zhang L. Xie J. et al.Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.Cancer Cell. 2012; 22: 585-600Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Previously, a chemical genomics screen identified the compound MJE3 as a non-specific target of PGAM1 that inhibits PGAM1 enzyme activity and breast cancer cell growth (Evans et al., 2005Evans M.J. Saghatelian A. Sorensen E.J. Cravatt B.F. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling.Nat. Biotechnol. 2005; 23: 1303-1307Crossref PubMed Scopus (195) Google Scholar). Detailed biochemical analysis revealed that Y26 phosphorylation of PGAM1 leads to increased activation by releasing E19 from the active site, which stabilizes binding of cofactor 2,3-bisphosphoglycerate and H11 phosphorylation (Hitosugi et al., 2013Hitosugi T. Zhou L. Fan J. Elf S. Zhang L. Xie J. Wang Y. Gu T.L. Alečković M. LeRoy G. et al.Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation.Nat. Commun. 2013; 4: 1790Crossref PubMed Scopus (70) Google Scholar). Recent studies have demonstrated that PGAM1 enzyme activity also has a non-metabolic role in homologous recombination (HR)-mediated DDR. PGAM1 enzyme activity regulates stability of CTBP-interacting protein (CtIP), a HR component that mediates replication protein A recruitment and Rad51 filament formation (Qu et al., 2017Qu J. Sun W. Zhong J. Lv H. Zhu M. Xu J. Jin N. Xie Z. Tan M. Lin S.H. et al.Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells.J. Cell Biol. 2017; 216: 409-424Crossref PubMed Scopus (43) Google Scholar). On the other hand, enzymatically dead PGAM1 demonstrated interesting non-metabolic function by promoting cancer metastasis. PGAM1 interacts with α-smooth muscle actin (ACTA2) and thereby modulates actin filaments assembly, cell motility, and cancer cell migration independent of its metabolic activity. Enzymatically inactive H186R mutant PGAM1 can bind ACTA2, whereas metabolically active, 201–210 amino acid-deleted PGAM1 cannot. In a xenograft model, decreased metastasis was observed with PGAM1 loss and was found to be prognostic in human breast cancer patients along with ACTA2 (Zhang et al., 2017Zhang D. Jin N. Sun W. Li X. Liu B. Xie Z. Qu J. Xu J. Yang X. Su Y. et al.Phosphoglycerate mutase 1 promotes cancer cell migration independent of its metabolic activity.Oncogene. 2017; 36: 2900-2909Crossref PubMed Scopus (57) Google Scholar). Here we report that PGAM1 is overexpressed in human gliomas across all grades (I–IV) and in established glioma cell lines. This overexpression of PGAM1 indirectly increases the efficiency of DDR and increases resistance to IR and temozolomide (TMZ) treatment. In addition, we show that PGAM1 traps WIP1 in the cytoplasm, thereby controlling phosphorylation of major cell cycle checkpoint proteins, such as ATM, Chk1, Chk2, and γ-H2AX. Together, these data suggest that PGAM1 inhibitors that block its interaction with WIP1 are ideal candidates to sensitize cells against DNA-damaging therapeutic agents. To determine if PGAM1 is expressed in primary brain tumors, we examined RNA levels of PGAM1 in human gliomas (grades I–IV) and normal brain obtained from frozen and formalin-fixed tissue samples. Real-time PCR gene expression analysis was performed with two independent sets of primers. As shown in Figure 1A, PGAM1 is overexpressed almost 4-fold in grade I tumors compared with normal brain, suggesting that overexpression of PGAM1 consistently occurs in gliomas from histologic grades I to IV. World Health Organization (WHO) grade III and IV gliomas express a small but statistically significant increase in PGAM1 expression compared with grade I tumors that is almost 5-fold overexpressed compared with normal brain. To find an in vitro model that resembles the PGAM1 overexpression found in human tumors, we examined three established glioma cell lines and compared them with normal human astrocytes (NHAs) using the same real-time PCR assay. Similar to primary human tumors, established glioma cell lines showed an almost 3.5- to 4-fold increase in PGAM1 expression compared with NHAs (Figure 1B). Similarly, The Cancer Genome Atlas (TCGA) dataset analyses demonstrated overexpression of PGAM1 compared with normal brain in grade II–IV gliomas (Figure 1C). To determine if the changes in PGAM1 expression noted at the RNA level were reflected in PGAM1 activity and protein expression, fixed material and lysates from frozen samples used for RNA analysis were subjected to a biochemical assay (to measure enzyme activity), western blot, and immunohistochemical analysis. As demonstrated by the biochemical assay, low-PGAM1-expressing normal brain samples exhibited low levels of PGAM1 enzyme activity, while the PGAM1 enzyme activity of the glioma samples correlated well with increased expression of the PGAM1 tumor mRNA (Figure 1D). As shown in the western blots in Figure 1E, representative normal brain samples expressed significantly less PGAM1 protein than brain tumor samples or commonly used glioma cell lines and three primary glioma stem-like cells (GSCs) established at the University of California, San Francisco (UCSF) (Fouse et al., 2014Fouse S.D. Nakamura J.L. James C.D. Chang S. Costello J.F. Response of primary glioblastoma cells to therapy is patient specific and independent of cancer stem cell phenotype.Neuro Oncol. 2014; 16: 361-371Crossref PubMed Scopus (25) Google Scholar, Mancini et al., 2018Mancini A. Xavier-Magalhães A. Woods W.S. Nguyen K.T. Amen A.M. Hayes J.L. Fellmann C. Gapinske M. McKinney A.M. Hong C. et al.Disruption of the β1L isoform of GABP reverses glioblastoma replicative immortality in a TERT promoter mutation-dependent manner.Cancer Cell. 2018; 34: 513-528Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar; Figure 1F). These results were consistent with immunohistochemical analyses of fixed tissue (Figure 1G), where semiquantitative analysis (Figure 1H) showed, as noted at the RNA level, that normal brain expresses low levels of PGAM1 protein compared with tumor samples. Upregulation of PGAM1 may be an early event in tumorigenesis, suggesting that PGAM1 is critical for glioma growth. To address this possibility, we modulated the expression of PGAM1 and monitored the effect on in vitro and in vivo growth of these cells. For these studies, U87, LN319, SF10602, and SF7996 glioma cells were used, as these cells demonstrate similar levels of PGAM1 mRNA and protein compared with human glioma samples (Figures 1B and 1F). Because all analyzed astrocytomas upregulate PGAM1, and because suppression of PGAM1 levels have been shown to inhibit tumor cell growth in other systems (Hitosugi et al., 2012Hitosugi T. Zhou L. Elf S. Fan J. Kang H.B. Seo J.H. Shan C. Dai Q. Zhang L. Xie J. et al.Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.Cancer Cell. 2012; 22: 585-600Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, Evans et al., 2005Evans M.J. Saghatelian A. Sorensen E.J. Cravatt B.F. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling.Nat. Biotechnol. 2005; 23: 1303-1307Crossref PubMed Scopus (195) Google Scholar), we first examined the consequences of PGAM1 knockdown in GBM cells. As shown in the western blot in Figure 2A, the lentiviral introduction of two different short hairpin RNAs (shRNAs) targeting PGAM1 resulted in two independent populations of GBM cells for each cell line, each of which exhibited significant decreases in PGAM1 expression compared with parental and empty vector controls. Decreased PGAM1 expression was associated with a significant decrease in PGAM1 activity (Figure 2B). In contrast to previously published data, PGAM1 suppression did not affect proliferation or colony formation (Figures 2C and 2D) in any of the glioma cell lines. Interestingly, when these PGAM1-knockdown cells were treated with IR and TMZ, they showed significantly increased sensitivity compared with the parental cells in terms of cell viability (Figures 2C and 2G) and apoptosis (Figures 2E and 2I; Figures S1A and S1B). Moreover, these cells formed significantly fewer colonies (Figures 2D and 2H) than the parental cells. However, in contrast to glioma cells, NHAs expressed low levels of PGAM1 and were more susceptible to both IR and TMZ, which did not further increase by PGAM1 silencing (Figures S1C and S1D), and western blot analysis demonstrated the presence of cleaved caspase-3 and cleaved PARP in PGAM1-knockdown cells treated with IR and TMZ, consistent with induction of apoptosis (Figures 2F and 2J). To mimic genetic loss, we used pharmacological inhibitors (PGAM-tide and Alizarin) that block PGAM1 enzyme activity (Hitosugi et al., 2012Hitosugi T. Zhou L. Elf S. Fan J. Kang H.B. Seo J.H. Shan C. Dai Q. Zhang L. Xie J. et al.Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.Cancer Cell. 2012; 22: 585-600Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, Engel et al., 2004Engel M. Mazurek S. Eigenbrodt E. Welter C. Phosphoglycerate mutase derived polypeptide inhibits glycolytic flux and induces cell growth arrest in tumor cell lines.J. Biol. Chem. 2004; 279: 35803-35812Crossref PubMed Scopus (57) Google Scholar; Figure S1E), but surprisingly this had no effect on cell viability or apoptosis when combined with IR (Figures S1F and S1G) and TMZ (Figures S1H and S1I) treatment. IR and TMZ treatment induce the formation of double-strand DNA breaks and activation of the DDR pathway. We therefore wanted to investigate if PGAM1 loss sensitizes GBM cells to IR and TMZ treatment through regulation of the DDR pathway. PGAM1-knockdown cells were subjected to IR and TMZ treatment, and double-strand breaks were measured using a single-cell comet assay performed under neutral pH conditions. In the control cells, fragmented DNA resulting from the double-strand breaks decreased over time, whereas fragmented DNA remained elevated in PGAM1-knockdown cells. After ionizing radiation treatment (10 Gy) in parental cells, the maximum number of double-strand breaks was found after 24 h and began gradually decreasing by 72 h. In PGAM1-knockdown cells, the number of double-strand DNA breaks remained unchanged from 24 h for up to 7 days (Figure 3A). Similarly, after TMZ (100 μM) treatment in parental cells, the maximum number of double-strand breaks was detected after 72 h and then gradually declined, whereas it remained elevated in PGAM1-knockdown cells for up to 7 days (Figure 3B). Previously identified PGAM1 interactors such as CtIP and ACTA2 had no significant effect on the extent of DNA damage or the repair of DNA breaks, as demonstrated by small interfering RNA (siRNA)-mediated knockdown followed by IR (10 Gy) treatment and comet assay (Figure S2A). But as expected, treatment with IR (10 Gy) resulted in formation of γ-H2AX foci (a surrogate measure of double-strand DNA breaks) in parental U87 and LN319 cells at day 1, which gradually decreased on days 5 and 7, indicating efficient repair of DNA damage (Figure 3C). Very few γ-H2AX foci were observed in U87 and LN319 PGAM1-knockdown cells after IR treatment, which did not change significantly over time (day 3 versus day 5 versus day 7) (Figure 3C). Similarly, 3 h exposure of parental cells to TMZ (100 μM) resulted in formation of γ-H2AX foci beginning 2 days after drug treatment, with a gradual decrease at days 5 and 7. Contrary to the parental cells, few γ-H2AX foci were observed in PGAM1-knockdown cells after TMZ treatment, which remained unchanged over time (Figure 3D). At early time points, DNA breaks are infrequent until 12 h (Figure S2B). Notably, NHAs treated with IR and TMZ showed significantly higher level of DNA breaks compared to the parental glioma cells and the DNA breaks in the NHAs were not similarly resolved with time (Figure S2C). PGAM1 knockdown in NHAs did not provide any significant additive changes in the extent of DNA breaks and γ-H2AX foci formation (Figures S2C and S2D). Response to DNA damage is related to DDR efficiency, and HR has previously been reported to be altered upon suppression of PGAM1 (Qu et al., 2017Qu J. Sun W. Zhong J. Lv H. Zhu M. Xu J. Jin N. Xie Z. Tan M. Lin S.H. et al.Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells.J. Cell Biol. 2017; 216: 409-424Crossref PubMed Scopus (43) Google Scholar). We therefore looked at HR and non-homologous end joining (NHEJ) efficiency by using a plasmid-based system using the ATM kinase inhibitor KU-55933 and DNA ligase-4 inhibitor SCR-7 as a positive control for HR and NHEJ inhibition. However, no change in HR or NHEJ efficiency was observed in parental or PGAM1-knockdown cells (Figure S2E), whereas KU-55933 and SCR-7 significantly lowered HR and NHEJ efficiency, respectively. This suggests that suppression of PGAM1 does not regulate HR and NHEJ efficiency in gliomas. ATM is one of the major kinases involved in the cellular response to DNA double-strand breaks, which may arise through the collapse of stalled replication forks or through exposure to DNA-damaging agents. Autophosphorylation of ser1981 is a reliable marker of ATM activation (Curtin, 2013Curtin N.J. Inhibiting the DNA damage response as a therapeutic manoeuvre in cancer.Br. J. Pharmacol. 2013; 169: 1745-1765Crossref PubMed Scopus (59) Google Scholar). We therefore treated U87 and LN319 cells with IR and TMZ, and performed western blotting to determine whether ATM phosphorylation would differ on the basis of PGAM1 expression. Phosphorylation of ATM ser1981 was readily observed in parental U87 and LN319 cells after both IR (10 Gy) and TMZ (100 μM) treatment as expected 48 h post-treatment (Figure 4A). At earlier time points before 48 h, no change in phosphorylation of ATM ser1981 was observed (Figure S3A). In contrast, phosphorylation of ATM ser1891 was not observed in PGAM1-knockdown U87 and LN319 cells after IR or TMZ treatment, indicating lack of ATM activation in these cell populations (Figure 4A). Moreover, in parental cells, IR treatment activated ATM and downstream signaling, as observed by phosphorylation of Chk2 at thr68 and cdc25C at ser216. However, phosphorylation of Chk2 thr68 or cdc25 ser216 was not observed in PGAM1-knockdown cells, which is consistent with deficient ATM signaling. TMZ treatment in parental cells resulted in phosphorylation of Chk1(ser345), Chk2(thr68), and cdc25C(ser216), which was not observed in PGAM1-knockdown cells. Consequently, as PGAM1-knockdown cells demonstrated loss of activating phosphorylation of Chk1 and Chk1, we found significantly increased G2/M arrest followed by apoptosis in response to IR and TMZ compared with corresponding parental cells (Figure 4B). To determine if PGAM1 knockdown affects the protein kinase ATR, an ATR kinase assay was performed on corresponding PGAM1-proficient and PGAM1-deficient cells. Phosphorylation of Chk1 in parental cells paralleled the activation of ATR, as demonstrated by the presence of serine/threonine phosphorylation of the ATR substrate PHAS by ATR immunoprecipitates of TMZ-treated cells. In contrast, in TMZ-treated PGAM1-knockdown cells, ATR activation was observed, but there was no activation of Chk1, as demonstrated by lack of Chk1 ser345 phosphorylation (Figure 4C). Our observations collectively indicate that PGAM1 expression is necessary for ATM downstream signaling in response to IR and TMZ in glioma cells. Because PGAM1 is mainly a cytoplasmic protein and may not be directly regulating dephosphorylation of nuclear DDR proteins, we next looked at proteins that shuttle between the cytoplasm and nucleus and play roles in regulating phosphorylation of DDR proteins. We selected five proteins (PP1, PP2A, PP4, PP6, and WIP1) and examined the effect of PGAM1 knockdown on the localization of these five proteins using immunofluorescence analysis. Only WIP1, which was located predominantly in the cytoplasm of control cells (Figure 4D), shifted to the nucleus in PGAM1-knockdown cells in response to DNA damage. The other four proteins (PP1, PP2A, PP4, and PP6) did not show any changes in their cellular localization due to loss of PGAM1 in either cell line (Figure 4D). Moreover, no change in subcellular localization or induction of PGAM1 protein expression was observed with IR or TMZ treatment (Figure 4E). These results therefore suggest that loss of PGAM1 expression allows increased export of WIP1 to the nucleus. Consequently, a shift in WIP1 localization from the cytoplasm to the nucleus was observed in PGAM1-knockdown cells using co-immunofluorescence analysis (Figure 4F). To determine the status of WIP1 protein expression in human glioma samples and cells lines, we studied the matched samples that have been used previously for PGAM1 expression analysis. A uniform WIP1 expression was detected in normal brain, normal astrocytes, glioma cell lines, and human glioma tumor samples (Figure S3B). Moreover, we analyzed WIP1 RNA expression using real-time PCR analysis and found no significant difference in RNA expression in gliomas compared with normal brain (Figure S3C). Similarly, U87 and LN319 expressed physiological levels of WIP1 (compared with the WIP1-overexpressing cell lines Lncap and MCF7), and there was no change in expression of WIP1 upon PGAM1 knockdown (Figure S3D). As WIP1 is a phosphatase found in the cytoplasm in the presence of PGAM1, we hypothesized that PGAM1 binds WIP1 to block WIP1-mediated interactions with ATM, Chk1, Chk2, and γ-H2AX. We therefore tested whether PGAM1 directly interacts with WIP1 to restrict movement toward the nucleus by performing immunoprecipitation and reverse immunoprecipitation using antibodies specific for PGAM1, WIP1, ATM, Chk1, Chk2, and γ-H2AX. In control cells, PGAM1 was detected in WIP1 immunoprecipitates, and WIP1 was detected in PGAM1 immunoprecipitates (Figure 5A; Figure S4A). Moreover, cell fractionation analysis revealed that PGAM1:WIP1 interaction is present only in the cytoplasmic fraction (Figure 5B), whereas no other phosphatases (PP1, PP2A, PP4, or PP6) were found to be immunoprecipitated with PGAM1 antibody (Figure S4B). In PGAM1-knockdown cells, PGAM1 was not detected in WIP1 immunoprecipitates, and WIP1 was accordingly not detected in PGAM1 immunoprecipitates (Figure 5C; Figures S4C and S4D). When the WIP1 antibody was used in PGAM1-knockdown cells, ATM, Chk1, Chk2, and γ-H2AX were all detected in WIP1 immunoprecipitates (Figure 5C; Figure S4D). We also performed immunoprecipitation using ATM, Chk1, Chk2, or γ-H2AX antibodies separately, and WIP1 was detected in each pull-down in PGAM1-knockdown cells (Figure 5C; Figure S4D). In each separate immunoprecipitation using a specific antibody targeting a DDR protein (ATM, Chk1, Chk2, or γ-H2AX), the other DDR proteins were not detected (Figure 5C; Figure S4D). Importantly, WIP1 was not observed in control U87 and LN319 cells immunoprecipitated with ATM, Chk1, Chk2, or γ-H2AX antibodies (Figure S4E). Loss of PGAM1 expression therefore allows WIP1 to associate with key nuclear signaling proteins in the DNA damage response pathway. To understand if the metabolic enzymatic activity of PGAM1 regulates the DDR pathway, we reconstituted a kinase-dead form of PGAM1 in stable PGAM1-knockdown cells (Figure 5D) and measured PGAM1 activity, DNA damage, and γ-H2AX foci formation in U87 and LN319 cells (Figures 5E–5G). As expected, the PGAM1 enzyme activity, 3PG production, and PPP flux in cells infected with two mutant (H186R and Y92F) PGAM1 constructs were similar to those of PGAM1-knockdown cells. In contrast, the enzyme activity (Figure 5E), 3PG production, and PPP flux (Figures S5A and S5B) in cells infected with a wild-type (WT) shRNA-resistant PGAM1 construct were similar to those in parental cells. Interestingly, reduction of γ-H2AX foci formation in PGAM1-knockdown cells was rescued by introduction of either the H186R or Y92F PGAM1 mutant or WT PGAM1 constructs (Figure 5F). As observed earlier, levels o" @default.
• W3016004654 created "2020-04-17" @default.
• W3016004654 creator A5009633669 @default.
• W3016004654 creator A5028321141 @default.
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• W3016004654 date "2020-04-01" @default.
• W3016004654 modified "2023-10-18" @default.
• W3016004654 title "Phosphoglycerate Mutase 1 Activates DNA Damage Repair via Regulation of WIP1 Activity" @default.
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• W3016004654 doi "https://doi.org/10.1016/j.celrep.2020.03.082" @default.
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