cGAS has a central role in STING activation after
cGAS has a central role in STING activation after DNA sensing, but it is unknown if most of the DNA sensors act independently or in cooperation with the cGAS-cGAMP pathway. This was addressed for IFI16 (Almine et al., 2017, Jønsson et al., 2017) and PQBP1 (Yoh et al., 2015) but remains to be tested for DNA-PK. Individual DNA sensors may share redundancy with each other but may be important for responding to specific types of DNA while avoiding self DNA. DNA-PK acts in a larger complex containing HEXIM1, NEAT1 long non-coding RNA, cGAS, and paraspeckle components (splicing factor, proline- and glutamine-rich [SFPQ]; MATRIN3; RNA binding protein 14 [RBM14]; paraspeckle component 1 [PSPC1]; and non-POU domain-containing octamer-binding protein [NONO]) (Morchikh et al., 2017). Upon stimulation of BHQ with ISD, DNA-PKcs was phosphorylated, paraspeckle proteins were released, STING was recruited, and IRF3 was activated, in a HEXIM1-dependent manner. The assembly of the complex, the precise function of each subunit, and whether they influence cGAS enzymatic activity are unknown. C4 and C16, being specific inhibitors, may be useful tools to investigate the role of DNA-PK.
DNA-PK can detect viruses other than VACV, including hepatitis B virus (Li et al., 2016), HSV-2 (Sui et al., 2017), and human T lymphotropic virus type 1 (Wang et al., 2017). Whether DNA-PK can sense other viruses and bacteria remains to be tested, and this testing should include nuclear DNA pathogens because DNA-PK is predominately nuclear. Other pathogens may have proteins to evade this receptor, yet VACV C4 and C16 are the only ones identified to date.
This paper describes the second DNA sensor inhibitor expressed by VACV, and, similar to the first, it targets DNA-PK to block DNA binding. Interestingly, VACV has evolved two proteins that inhibit DNA-PK; however, it is not known to encode proteins that directly inhibit any of the other reported DNA sensors. This emphasizes the importance of DNA-PK as a PRR. Learning more about the C4 and C16 interactions with Ku may allow for the development of small molecule inhibitors that mimic this interaction. This would be relevant to human autoimmune diseases associated with dysregulated DNA sensing, for example Aicardi-Goutières (Ahn and Barber, 2014). Our work on the viral inhibitors C4 and C16, along with similar studies, contributes to the understanding of the complex initial stages of DNA detection and the interplay between the DNA damage response and inflammation.
Acknowledgments The authors thank Prof. Paul Hasty for supplying Xrcc5/Tp53 MEFs and Prof. Penelope Jeggo for providing Prkdc MEFs. We also thank Brian Ferguson for useful discussion regarding the DNA sensing ELISA assays. This work was funded by a grant from the Wellcome Trust. G.L.S. is a Wellcome Trust Principal Research Fellow, S.R.S. was supported by a Research Studentship from the Department of Pathology, University of Cambridge, and C.Y. was supported by a summer studentship from the Lister Institute.
Introduction Acute lymphoblastic leukemia (ALL) is the most common malignancy in children accounting for almost 30% of pediatric cancers and 80% of childhood leukemias. Despite long-term survival and high cure rates for the majority of children diagnosed with ALL, disease relapse and resistance to treatment is the leading cause of death in pediatric ALL . Therefore, it is of great importance to identify novel therapeutic approaches for improvement in overall survival of the patients. Current treatment for ALL includes combination of chemotherapy drugs such as vincristine, steroids, asparaginase, and an anthracycline . Doxorubicin is one of the most commonly used anthracycline chemotherapeutic drugs that intercalates between DNA bases, inhibits topoisomerase II enzyme and subsequently induces DNA double strand breaks (DSBs) in tumor cells . However, increased DSB repair activity as observed in many cancers, can render tumor cells resistant to topoisomerase II inhibitors like doxorubicin . In mammalian cells, repair of DNA DSBs mediated by two main pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). NHEJ is considered as error-prone repair pathway with the potential to ligate any kind of DSB ends without requirement for a homologous sequence as a template . A growing body of evidence supports a major role for NHEJ in conferring resistance to cancer cells against radio- and chemotherapeutic agents that damage DNA , . Importantly, it has been demonstrated that up-regulation of NHEJ family genes correlates with relapse in pediatric ALL . Therefore, targeting NHEJ processing enzymes is likely to have a profound impact on the efficacy of DNA damaging agents . DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of NHEJ pathway of DSB repair . It is known that the kinase activity of DNA-PKcs is required for the repair of DSBs by NHEJ and inhibition of DNA-PKcs phosphorylation associates with deficient DSB repair in response to DNA damaging agents . NU7441 was identified as a potent and specific DNA-PK inhibitor which increases the sensitivity of colon and breast cancer cells to ionizing radiation and topoisomerase II inhibitors , . Furthermore, inhibition of DNA-PK using NU7441 sensitizes chronic lymphocytic leukemia cells to chemotherapeutic compounds. However, the effects of DNA-PK inhibition in the presence of DSB-inducing drug has not yet been elucidated in B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. In the present study, we investigated the effects of DNA-PK inhibition by NU7441 on doxorubicin-induced DSBs in BCP-ALL cells.