DNA-dependent Protein Kinase In Response Research Articles

UbcH5c-dependent activation of DNA-dependent protein kinase in response to replication-mediated DNA double-strand breaks

Camptothecin (CPT) exhibits strong cytotoxicity by inducing DNA double-strand breaks (DSBs) through DNA replication. Unlike radiation-induced DSBs, which have two DNA ends, CPT-induced DSBs are considered to have only one DNA end. However, the differences in cellular responses to one-ended and two-ended DSBs are not well understood. Our previous study showed that proteasome inhibitor treatment suppressed CPT-induced activation of DNA-PK, a factor required for non-homologous end-joining in DSB repair, suggesting that the ubiquitin-proteasome pathway is involved in DNA-PK activation in response to one-ended DSBs. To identify the ubiquitination factors required for DNA-PK activation, we screened an siRNA library against E2 ubiquitin-conjugating enzymes and identified UbcH5c. Knockdown of UbcH5c suppressed DNA-PK activation caused by CPT, but not by the radio-mimetic drug neocarzinostatin. UbcH5c-dependent DNA-PK activation occurred independent of DNA end resection. Furthermore, loss of UbcH5c reduced DNA-PK-dependent chromosomal aberrations and suppressed the activation of cell cycle checkpoint in response to CPT. These results suggest that UbcH5c regulates DNA-PK activation in response to one-ended DSBs caused by replication fork collapse. To our knowledge, this is the first report of a DSB repair-related factor that is differentially involved in the response to one- and two-ended DSBs.

PGC1α Degradation Suppresses Mitochondrial Biogenesis to Confer Radiation Resistance in Glioma.

Glioma cells reduce mitochondrial biogenesis by promoting PGC1α degradation to promote resistance to radiotherapy, indicating potential therapeutic strategies to enhance radiosensitivity.

Abstract 271: The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells

Abstract Therapeutic targeting of the DNA damage response (DDR) has the potential to improve the poor survival outcomes of pancreatic ductal adenocarcinoma (PDAC). Many common genetic alterations in PDAC augment replication stress, which activates Ataxia-telangiectasia and Rad3-related kinase (ATR). We have demonstrated previously, efficacy of the ATR inhibitor (AZD6738) in PDAC models, particularly when combined with gemcitabine (gem) (Wallez et al, MCT, 2018). This combination strongly induces activation of Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), indicating the induction of double-strand breaks. It has been suggested that ATM-deficiency can sensitize cancer cells to ATR inhibitors. This study sought to assess the relevance of this finding to PDAC and to interrogate the distinct roles of ATM and DNA-PK in response to ATRi/gem in PDAC cell lines. The ATM inhibitor, AZD0156, sensitized human pancreatic cancer cell lines (MIA PaCa-2, HPAF-II, AsPC-1) to AZD6738, as determined by SRB assays (e.g. MIA PaCa-2; AZD6738 GI50 = 3.8 µM, versus 1.3 µM in the presence of 30 nM AZD0156) and in longer term colony forming assays (surviving fraction (SF) (1 µM AZD6738); 78 +/- 0.9%, SF (1 µM AZD6738 + 30nM AZD0156); 11 +/- 4.2%). However, ATM knockdown with siRNA did not sensitize to AZD6738, nor to the combination of AZD6738/gem. This suggests that the presence of kinase-inhibited ATM at sites of DNA damage is more deleterious to PDAC cells than deficiency of ATM protein expression. We established that AZD6738/gem could induce phosphorylation of the canonical targets of ATM (Chk2, KAP1) in MIA PaCa-2 when ATM was knocked down or inhibited. DNA-PK was also activated in response to exposure to ATRi/gem and we postulated that it was responsible for the Chk2/KAP1 activation. We then demonstrated that Chk2/KAP1 activation could be abrogated by DNA-PK inhibition with NU7441. Furthermore, in an ATM-null pancreatic cancer cell line, AZD6738/gem-induced KAP1 phosphorylation was also abrogated by inhibition of DNA-PK. Thus, DNA-PK appears to be responsible for the downstream activation of Chk2 and KAP1, induced by AZD6738/gem in PDAC cells. Therefore, as well as revealing that inhibition of ATM using the novel inhibitor AZD0156 is highly deleterious to ATR inhibited PDAC cells, we have identified a compensatory mechanism via DNA-PK, which may be relevant for maximizing the therapeutic potential of DDR inhibitors in PDAC. Since DNA-PK clearly plays a dominant role in the DDR signaling pathways, we are now assessing whether DNA-PK inhibition synergizes with AZD6738+/- gem. Citation Format: Charles R. Dunlop, Yann Wallez, Sandra Bernaldo de Quirós Fernández, Saadia A. Karim, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 271.

In cellulo phosphorylation of DNA double-strand break repair protein XRCC4 on Ser260 by DNA-PK.

XRCC4 is one of the core factors for DNA double-strand break (DSB) repair through non-homologous end joining (NHEJ). XRCC4 is phosphorylated by DNA-dependent protein kinase (DNA-PK), with Ser260 and Ser320 (Ser318 in the alternatively spliced form) being the major phosphorylation sites in vitro. It was recently reported that Ser320 is phosphorylated by DNA-PK in response to DNA damage; however, it is currently unclear whether Ser260 is phosphorylated in cellulo in response to DNA damage. Herein, we generated an antibody against XRCC4 phosphorylated on Ser260 and examined its phosphorylation status via Western blotting. XRCC4 Ser260 phosphorylation increased after irradiation with 30–300 Gy of γ-rays and was suppressed by DNA-PK inhibitor but not by ATM inhibitor. Moreover, XRCC4 Ser260 phosphorylation decreased in DNA-PKcs–deficient cells. These observations indicate that XRCC4 Ser260 is phosphorylated by DNA-PK in cellulo. The XRCC4S260A mutant reversed the high radiosensitivity of XRCC4-deficient M10 cells to a similar level to that of wild-type XRCC4. However, the clonogenic survival of cells expressing the XRCC4S260A mutant was slightly but significantly lower than that of those expressing wild-type XRCC4. In addition, XRCC4S260A-expressing cells displayed a significantly greater number of γ-H2AX foci than XRCC4WT-expressing cells 4 h after 1 Gy irradiation and without irradiation. The present results suggest a potential role of XRCC4 Ser260 phosphorylation by DNA-PK in DSB repair.

Induction of H2AX phosphorylation in tumor cells by gossypol acetic acid is mediated by phosphatidylinositol 3-kinase (PI3K) family.

BackgroundH2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including Ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. Our study shows that gossypol acetic acid (GAA) alone can induce γH2AX in Human mucoepidermoid carcinoma cell line (MEC-1) in vitro. Thus, we further examined the possible mechanisms of GAA to induce γH2AX in tumor cells.Materials and methodsThe PI3K inhibitors caffeine and wortmannin were used in an effort to identify the kinase(s) responsible for GAA -induced γH2AX in MEC-1 cells. DNA dependent protein kinase (DNA-PK) - proficient and –deficient cells, human glioma cell lines M059K and M059J, were also used to evaluate the kinases responsible for GAA induced H2AX phosphorylation. γH2AX expression was detected by immunofluorescent microscopy. Flow cytometry assay was used to assay γH2AX and cell cycle.ResultsGAA induced H2AX phosphorylation in a cell cycle-dependent manner and a significant G0/G1 phase arrest in MEC-1 cells was shown. Caffeine and wortmannin significantly inhibited GAA-induced H2AX phosphorylation in MEC-1 cells. GAA induced H2AX phosphorylation in M059K, but not in M059J. Taken together, these data suggested that GAA treatment alone could induce H2AX phosphorylation in a cell cycle dependent manner in MEC-1 and M059K, but not in M059J cells. A significant G0/G1 phase arrest was shown in MEC-1.ConclusionsThe member of PI3K family, DNA-PK, ATM and ATR are involved in the H2AX phosphorylation of MEC-1 cells.

Abstract 3329: The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells.

Abstract Microtubules are involved in the formation of the mitotic spindle, and correct separation of the sister chromatids at mitosis is essential for maintaining genomic stability. Microtubule-targeting drugs arrest rapidly-dividing cells and are important chemotherapeutic agents, but the development of multidrug-resistance (MDR) is a serious clinical problem. Along with its role in DNA double-strand break repair, DNA-dependent protein kinase (DNA-PK) has also been shown to play an important role in stabilising spindle formation and preventing mitotic catastrophe (Shang et al, Cancer Res. 2010). In light of this novel finding, we hypothesised that inhibition of DNA-PK would sensitise cells to microtubule-targeting agents. Pilot studies showed that inhibition of DNA-PK by the selective inhibitor, NU7441, caused greater sensitisation to vincristine in multidrug-resistant cells compared to parental cells. Here, we further investigate the role of DNA-PK in response to microtubule-targeting drugs using a novel series of NU7441 analogues, and investigate whether these inhibitors interact with the efflux transporter, P-glycoprotein. In growth inhibition studies, CCRF-CEM leukaemia cells were sensitised 1.4-fold to vincristine by NU7441 (1μM) whereas their vincristine-resistant derivative CCRF-CEM VCR/R cells were sensitised 8-fold to vincristine. DNA-PK-deficient M059J glioblastoma cells were 2-fold more sensitive to docetaxel than DNA-PK-proficient cells, and NU7441 sensitised only DNA-PK-proficient M059J-Fus1 cells to docetaxel. CCRF-CEM VCR/R cells have a higher basal level of autophosphorylation of DNA-PKcs (Western blotting) than the CCRF-CEM cells and autophosphorylation in response to vincristine was abrogated by NU7441. To investigate the effects of NU7441 on drug transport, a doxorubicin fluorescence assay showed that in P-glycoprotein-overexpressing canine kidney MDCKII-MDR1 cells, 1μM NU7441 increased doxorubicin cellular fluorescence 16-fold by blocking drug efflux via P-glycoprotein. NU7441 and 3 structurally-related compounds (NU7742 (inactive NU7441 analogue), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative) or DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC-MS. However, the non-inhibitory compound NU7742 was 5-fold less effective at sensitising CCRF-CEM VCR/R cells to vincristine than NU7441 (p=0.008). The novel finding that DNA-PK inhibition sensitises cells to microtubule-targeting agents suggests that along with its function in DNA repair, this enzyme plays a role in the mitotic spindle checkpoint. Additionally, in multidrug-resistant cells, NU7441 is able to block drug efflux via interaction with P-glycoprotein. Citation Format: Emily Mould, Philip Berry, David Jamieson, Christopher Hill, Niu Tan, Sarah Elliott, Barbara Durkacz, David Newell, Elaine Willmore. The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2013-3329

Vaccinia-related Kinase 1 (VRK1) Is an Upstream Nucleosomal Kinase Required for the Assembly of 53BP1 Foci in Response to Ionizing Radiation-induced DNA Damage

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.

Activation of DNA-PK by Ionizing Radiation Is Mediated by Protein Phosphatase 6

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous end-joining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1γ1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.

XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage

The two BRCT domains (BRCT1 and BRCT2) of XRCC1 mediate a network of protein–protein interactions with several key factors of the DNA single-strand breaks (SSBs) and base damage repair pathways. BRCT1 is required for the immediate poly(ADP–ribose)-dependent recruitment of XRCC1 to DNA breaks and is essential for survival after DNA damage. To better understand the biological role of XRCC1 in the processing of DNA ends, a search for the BRCT1 domain-associated proteins was performed by mass spectrometry of GST-BRCT1 pulled-down proteins from HeLa cell extracts. Here, we report that the double-strand break (DSB) repair heterotrimeric complex DNA-PK interacts with the BRCT1 domain of XRCC1 and phosphorylates this domain at serine 371 after ionizing irradiation. This caused XRCC1 dimer dissociation. The XRCC1 R399Q variant allele did not affect this phosphorylation. We also show that XRCC1 strongly stimulates the phosphorylation of p53-Ser15 by DNA-PK. The pseudo phosphorylated S371D mutant was a much weaker stimulator of DNA-PK activity whereas the non-phosphorylable mutant S371L endowed with a DNA-PK stimulating capacity failed to fully rescue the DSB repair defect of XRCC1-deficient EM9 rodent cells. The functional association between XRCC1 and DNA-PK in response to IR provides the first evidence for their involvement in a common DSB repair pathway.

Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase.

Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein. We report here that HSF1 binds specifically to each of the two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer. The binding is blocked by antibodies against HSF1. Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress.

Functional interaction between DNA-PK and c-Abl in response to DNA damage.

How DNA damage is converted into intracellular signals that can control cell behaviour is unknown. The c-Abl protein tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of a serine/threonine kinase and Ku DNA-binding subunits, requires DNA double-strand breaks or other DNA lesions for activation. Here we demonstrate that c-Abl interacts constitutively with DNA-PK. Ionizing radiation stimulates binding of c-Abl to DNA-PK and induces an association of c-Abl with Ku antigen. We show that DNA-PK phosphorylates and activates c-Abl in vitro. Cells deficient in DNA-PK are defective in c-Abl activation induced by ionizing radiation. In a potential feedback mechanism, c-Abl phosphorylates DNA-PK, but not Ku, in vitro. Phosphorylation of DNA-PK by c-Abl inhibits the ability of DNA-PK to form a complex with DNA. We also show that treatment of cells with ionizing radiation results in phosphorylation of DNA-PK that is dependent on c-Abl. Our results support the hypothesis that there are functional interactions between c-Abl and DNA-PK in the response to DNA damage.