TOPK Inhibition Enhances the Sensitivity of Colorectal Cancer Cells to Radiotherapy by Reducing the DNA Damage Response

Objective Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase(TOPK)was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer.However,the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer(CRC)cells is unclear.This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells.Methods The expression of TOPK was detected in CRC tissues by immunohistochemistry,and the effect of TOPK knockdown was detected in CRC cells by Western blotting.CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells.Furthermore,proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy.DNA damage was detected by the comet assay.Changes in the DNA damage response signaling pathway were analyzed by Western blotting,and apoptosis was detected by flow cytometry.Results The expression of TOPK was significantly greater in CRC tissues at grades 2–4 than in those at grade 1.After irradiation,CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins,including phospho-cyclin-dependent kinase 1(p-CDK1),phospho-ataxia telangiectasia-mutated(p-ATM),poly ADP-ribose polymerase(PARP),and meiotic recombination 11 homolog 1(MRE11).Conclusions TOPK was overexpressed in patients with moderately to poorly differentiated CRC.Moreover,TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.

Poly(ADP-ribosyl)ation of Apoptosis Antagonizing Transcription Factor Involved in Hydroquinone-Induced DNA Damage Response

The molecular mechanism of DNA damage induced by hydroquinone （HQ） remains unclear. Poly（ADP-ribose） polymerase-1 （PARP-1） usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor （AATF）, PARP-1, and phosphorylated H2AX （v-H2AX） were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-l-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, y-H2AX expression increased to 265%. Co-immunoprecipitation （co-IP） assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly（ADP-ribosyl）ation {PARylation） regulated AATF expression, in conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.

Decoupling of DNA damage response signaling from DNA damages underlies temozolomide resistance in glioblastoma cells

Glioblastoma multiforme （GBM） is the most aggressive primary brain tumor in adults.Current therapy includes surgery,radiation and chemotherapy with temozolomide （TMZ）.Major determinants of clinical response to TMZ include methylation status of the O6-methylguanine-DNA methyltransferase （MGMT） promoter and mismatch repair （MMR） status.Though the MGMT promoter is methylated in 45% of cases,for the first nine months of follow-up,TMZ does not change survival outcome.Furthermore,MMR deficiency makes little contribution to clinical resistance,suggesting that there exist unrecognized mechanisms of resistance.We generated paired GBM cell lines whose resistance was attributed to neither MGMT nor MMR.We show that,responding to TMZ,these cells exhibit a decoupling of DNA damage response （DDR） from ongoing DNA damages.They display methylation-resistant synthesis in which ongoing DNA synthesis is not inhibited.They are also defective in the activation of the S and G2 phase checkpoint.DDR proteins ATM,Chk2,MDC1,NBS1 and gammaH2AX also fail to form discrete foci.These results demonstrate that failure of DDR may play an active role in chemoresistance to TMZ.DNA damages by TMZ are repaired by MMR proteins in a futile,reiterative process,which activates DDR signaling network that ultimately leads to the onset of cell death.GBM cells may survive genetic insults in the absence of DDR.We anticipate that our findings will lead to more studies that seek to further define the role of DDR in ultimately determining the fate of a tumor cell in response to TMZ and other DNA methylators.

RECQL4 regulates DNA damage response and redox homeostasis in esophageal cancer

Objective:RECQL4(a member of the RECQ helicase family)upregulation has been reported to be associated with tumor progression in several malignancies.However,whether RECQL4 sustains esophageal squamous cell carcinoma(ESCC)has not been elucidated.In this study,we determined the functional role for RECQL4 in ESCC progression.Methods:RECQL4 expression in clinical samples of ESCC was examined by immunohistochemistry.Cell proliferation,cellular senescence,the epithelial-mesenchymal transition(EMT),DNA damage,and reactive oxygen species in ESCC cell lines with RECQL4 depletion or overexpression were analyzed.The levels of proteins involved in the DNA damage response(DDR),cell cycle progression,survival,and the EMT were determined by Western blot analyses.Results:RECQL4 was highly expressed in tumor tissues when compared to adjacent non-tumor tissues in ESCC(P<0.001)and positively correlated with poor differentiation(P=0.011),enhanced invasion(P=0.033),and metastasis(P=0.048).RECQL4 was positively associated with proliferation and migration in ESCC cells.Depletion of RECQL4 also inhibited growth of tumor xenografts in vivo.RECQL4 depletion induced G0/G1 phase arrest and cellular senescence.Importantly,the levels of DNA damage and reactive oxygen species were increased when RECQL4 was depleted.DDR,as measured by the activation of ATM,ATR,CHK1,and CHK2,was impaired.RECQL4 was also shown to promote the activation of AKT,ERK,and NF-k B in ESCC cells.Conclusions:The results indicated that RECQL4 was highly expressed in ESCC and played critical roles in the regulation of DDR,redox homeostasis,and cell survival.

DNA Damage Response in Resting and Proliferating Peripheral Blood Lymphocytes Treated by Camptothecin or X-ray

DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.

Evaluation of 30 DNA damage response and 6 mismatch repair gene mutations as biomarkers for immunotherapy outcomes across multiple solid tumor types

Objective:DNA damage response(DDR)genes have low mutation rates,which may restrict their clinical applications in predicting the outcomes of immune checkpoint inhibitor(ICI)treatment.Thus,a systemic analysis of multiple DDR genes is needed to identify potential biomarkers of ICI efficacy.Methods:A total of 39,631 patients with mutation data were selected from the cBioPortal database.A total of 155 patients with mutation data were obtained from the Fudan University Shanghai Cancer Center(FUSCC).A total of 1,660 patients from the MSK-IMPACT cohort who underwent ICI treatment were selected for survival analysis.A total of 249 patients who underwent ICI treatment from the Dana-Farber Cancer Institute(DFCI)cohort were obtained from a published dataset.The Cancer Genome Atlas(TCGA)level 3 RNA-Seq version 2 RSEM data for gastric cancer were downloaded from cBioPortal.Results:Six MMR and 30 DDR genes were included in this study.Six MMR and 20 DDR gene mutations were found to predict the therapeutic efficacy of ICI,and most of them predicted the therapeutic efficacy of ICI,in a manner dependent on TMB,except for 4 combined DDR gene mutations,which were associated with the therapeutic efficacy of ICI independently of the TMB.Single MMR/DDR genes showed low mutation rates;however,the mutation rate of all the MMR/DDR genes associated with the therapeutic efficacy of ICI was relatively high,reaching 10%–30%in several cancer types.Conclusions:Coanalysis of multiple MMR/DDR mutations aids in selecting patients who are potential candidates for immunotherapy.

Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage

Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） induced formation of topoisomerase I cleavable complex （TOPlcc）, degradation of TOP1 and activation of DNA damage responses （DDR）. Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPTo indneed TOPl degradation, while none of above three processing activities affected TOPlcc formation. Replication- and transcription-initiated proeessing （RIP and TIP） of TOPlee were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOPlec triggered the CPT-induced RPA pbosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chkl/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOPlcc-activated, but not ionization radiationactivated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respec- tively. Together, our results support that both RIP and TIP pathways of TOPlcc are required for the activation of CPT-induced DDR and cytotoxicity.

Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80

Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.

cGAS regulates the DNA damage response to maintain proliferative signaling in gastric cancer cells

The activation of some oncogenes promote cancer cell proliferation and growth,facilitate cancer progression and metastasis by induce DNA replication stress,even genome instability.Activation of the cyclic GMP-AMP synthase(cGAS)mediates classical DNA sensing,is involved in genome instability,and is linked to various tumor development or therapy.However,the function of cGAS in gastric cancer remains elusive.In this study,the TCGA database and retrospective immunohistochemical analyses revealed substantially high cGAS expression in gastric cancer tissues and cell lines.By employing cGAS high-expression gastric cancer cell lines,including AGS and MKN45,ectopic silencing of cGAS caused a significant reduction in the proliferation of the cells,tumor growth,and mass in xenograft mice.Mechanistically,database analysis predicted a possible involvement of cGAS in the DNA damage response(DDR),further data through cells revealed protein interactions of the cGAS and MRE11-RAD50-NBN(MRN)complex,which activated cell cycle checkpoints,even increased genome instability in gastric cancer cells,thereby contributing to gastric cancer progression and sensitivity to treatment with DNA damaging agents.Furthermore,the upregulation of cGAS significantly exacerbated the prognosis of gastric cancer patients while improving radiotherapeutic outcomes.Therefore,we concluded that cGAS is involved in gastric cancer progression by fueling genome instability,implying that intervening in the cGAS pathway could be a practicable therapeutic approach for gastric cancer.

New insight into targeting the DNA damage response in the treatment of glioblastoma

Glioblastoma(GBM)is the most common invasive malignant tumor in human brain tumors,representing the most severe grade of gliomas.Despite existing therapeutic approaches,patient prognosis remains dismal,necessitating the exploration of novel strategies to enhance treatment efficacy and extend survival.Due to the restrictive nature of the blood-brain barrier(BBB),smallmolecule inhibitors are prioritized in the treatment of central nervous system tumors.Among these,DNA damage response(DDR)inhibitors have garnered significant attention due to their potent therapeutic potential across various malignancies.This review provides a detailed analysis of DDR pathways as therapeutic targets in GBM,summarizes recent advancements,therapeutic strategies,and ongoing clinical trials,and offers perspectives on future directions in this rapidly evolving field.The goal is to present a comprehensive outlook on the potential of DDR inhibitors in improving GBM management and outcomes.

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

AIM： To explore whether acute cellular DNA damage response is induced upon hepatitis B virus （HBV） infection and the effects of the HBV infection. METHODS： We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chkl phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet （UV） radiation and ionizing radiation （IR）-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. RESULTS： We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chkl, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h aEer HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increasedapoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION： HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

The DNA damage and regulatory strategy in hematopoietic stem cells after irradiation exposure:Progress and challenges

The hematopoietic system is susceptible to ionizing radiation(IR),which can cause acute hematopoietic failure or long-term myelosuppression.As the most primitive cells of the hematopoietic hierarchy,hematopoietic stem cells(HSCs)maintain lifelong hematopoietic homeostasis and promote hematopoietic regeneration during stress.Numerous studies have shown that nuclear and mitochondrial genomes are the main targets of radiation injury in HSCs.More importantly,the damage of DNA may trigger a series of biological responses that largely determine HSC fate following IR exposure.Although some essential pathways and factors involved in DNA injury and damage in HSCs have been revealed,a comprehensive understanding of the biological effects of radiation on HSCs still needs to be improved.This review focuses on recent insights into the molecular mechanisms underlying DNA damage and repair in HSCs after IR.Then summarize corresponding regulatory measures,which may provide a reference for further research in this field.

New insights into ATR inhibition in muscle invasive bladder cancer:The role of apolipoprotein B mRNA editing catalytic subunit 3B

Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

Histone post-translational modification and the DNA damage response

DNA is highly vulnerable to spontaneous and environmental timely damage in living cells.DNA damage may cause genetic instability and increase cancer risk if the damages are not repaired timely and efficiently.Human cells possess several DNA damage response(DDR)mechanisms to protect the integrity of their genome.Clarification of the mechanisms under-lying the DNA damage response following lethal damage will facilitate the identification of therapeutic targets for cancers.Histone post-translational modifications(PTMs)have been indicated to play different roles in the repair of DNA damage.In this context,histone PTMs regulate recruitment of downstream effectors,and facilitate appropriate repair response.This review outlines the current understanding of different histone PTMs in response to DNA dam-age repair,besides,enumerates the role of new type PTMs such as histone succinylation and crotonylation in regulating DNA damage repair processes.

Hmo1:A versatile member of the high mobility group box family of chromosomal architecture proteins

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but also hinders DNA transactions.Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions.The high mobility group box(HMGB)proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion.They play a major role in chromatin dynamics.The Saccharomyces cerevisiae(yeast hereafter)HMGB protein Hmo1 contains two HMGB motifs.However,unlike a canonical HMGB protein that has an acidic C-terminus,Hmo1 ends with a lysine rich,basic,C-terminus,resembling linker histone H1.Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions.For instance,Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones.Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome.This minireview reviews the functions of Hmo1 and the underlying mechanisms,highlighting recent discoveries.

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.

Cellular DNA repair cofactors affecting hepatitis B virus infection and replication

AIM： To investigate whether hepatitis B virus （HBV） infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication. METHODS： Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated （ATM）- Rad3-related protein （ATR）, p21 and the level of phosphorylation of Chkl, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATN chemical inhibitors caffeine （CF） and theophylline （TP）, or Chkl inhibitor 7-hydroxystaurosporine （UCN01） was studied to determine whether they suppress cellular DNA damage response and NG132 inhibits proteasome. RESULTS： The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after NG132 treatment also sharply decreased the HBV DNA yield. CONCLUSION： HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatment of HBV infection.

A brief history of the DNA repair field

The history of the repair of damaged DNA can be traced to the mid-1930s. Since then multiple DNA repair mechanisms, as well as other biological responses to DNA damage, have been discovered and their regulation has been studied. This article briefly recounts the early history of this field.

TBC1D15 deficiency protects against doxorubicin cardiotoxicity via inhibiting DNAPKcs cytosolic retention and DNA damage

Clinical application of doxorubicin(DOX)is heavily hindered by DOX cardiotoxicity.Several theories were postulated for DOX cardiotoxicity including DNA damage and DNA damage response(DDR),although the mechanism(s)involved remains to be elucidated.This study evaluated the potential role of TBC domain family member 15(TBC1D15)in DOX cardiotoxicity.Tamoxifen-induced cardiac-specific Tbcldi5 knockout(Tbcldi5^(CKO))or Tbcldi5 knockin(Tbcldi5^(CKI))male mice were challenged with a single dose of DOx prior to cardiac assessment 1 week or 4 weeks following DOX challenge.Adenoviruses encoding TBC1D15 or containing shRNA targeting Tbcld15 were used for Tbcld15 overexpression or knockdown in isolated primary mouse cardiomyocytes.Our results re-vealed that DOX evoked upregulation of TBC1D15 with compromised myocardial function and overt mortality,the effects of which were ameliorated and accentuated by Tbcldi5 deletion and Tbcld15 overexpression,respectively.DOX overtly evoked apoptotic cell death,the effect of which was alleviated and exacerbated by Tbcld15 knockout and overexpression,respectively.Meanwhile,DOX provoked mitochondrial membrane potential collapse,oxidative stress and DNA damage,the effects of which were mitigated and exacerbated by Tbcld15 knockdown and overexpression,respectively.Further scrutiny revealed that TBC1D15 fostered cytosolic accumulation of the cardinal DDR element DNA-dependent protein kinase catalytic subunit(DNA-PKcs).Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation denoted an interaction between TBCID15 and DNA-PKcs at the segment 594-624 of TBC1D15.Moreover,overexpression of TBC1D15 mutant(A594-624,deletion of segment 594-624)failed to elicit accentuation of DOX-induced cytosolic retention of DNA-PKcs,DNA damage and cardiomyocyte apoptosis by TBC1D15 wild type.However,Tbcld15 deletion ameliorated DOXinduced cardiomyocyte contractile anomalies,apoptosis,mitochondrial anomalies,DNA damage and cytosolic DNA-PKcs accumulation,which were canceled off by DNA-PKcs inhibition or ATM activation.Taken together,our findings denoted a pivotal role for TBCID15 in DOX-induced DNA damage,mitochondrial injury,and apoptosis possibly through binding with DNA-PKcs and thus gate-keeping its cytosolic retention,a route to accentuation of cardiac contractile dysfunction in DOX-induced cardiotoxicity.

Alterations of DNA damage response pathway:Biomarker and therapeutic strategy for cancer immunotherapy

Genomic instability remains an enabling feature of cancer and promotes malignant transformation.Alterations of DNA damage response(DDR)pathways allow genomic instability,generate neoantigens,upregulate the expression of programmed death ligand 1(PD-L1)and interact with signaling such as cyclic GMPe AMP synthase-stimulator of interferon genes(cGASe STING)signaling.Here,we review the basic knowledge of DDR pathways,mechanisms of genomic instability induced by DDR alterations,impacts of DDR alterations on immune system,and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.