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. Conclusion: 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.

Synthesis and new DNA targeting activity of 6- and 7-tert-butylfascaplysins.

Fascaplysin is a red cytotoxic pigment with anticancer properties isolated from the marine sponge Fascaplysinopsis sp. Recently, structure-activity relationship analysis reported by our group suggested that selective cytotoxicity of fascaplysin derivatives towards tumor cells negatively correlates with their ability to intercalate into DNA. To validate this hypothesis, we synthesized 6- and 7-tert-butylfascaplysins which reveal mitigated DNA-intercalating properties. These derivatives were found to be strongly cytotoxic to drug-resistant human prostate cancer cells, albeit did not demonstrate improved selectivity towards cancer cells when compared to fascaplysin. At the same time, kinome analysis suggested an activation of CHK1/ATR axis in cancer cells shortly after the drug exposure. Further experiments revealed induction of replication stress that is eventually converted to the toxic DNA double-strand breaks, resulting in caspase-independent apoptosis-like cell death. Our observations highlight new DNA-targeting effect of some fascaplysin derivatives and indicate more complex structure-activity relationships within the fascaplysin family, suggesting that cytotoxicity and selectivity of these alkaloids are influenced by multiple factors. Furthermore, combination with clinically-approved inhibitors of ATR/CHK1 as well as testing in tumors particularly sensitive to the DNA damage should be considered in further studies.

Uncovering miRNA-mRNA Regulatory Networks Related to Olaparib Resistance and Resensitization of BRCA2MUT Ovarian Cancer PEO1-OR Cells with the ATR/CHK1 Pathway Inhibitors.

Resistance to olaparib is the major obstacle in targeted therapy for ovarian cancer (OC) with poly(ADP-ribose) polymerase inhibitors (PARPis), prompting studies on novel combination therapies to enhance olaparib efficacy. Despite identifying various mechanisms, understanding how OC cells acquire PARPi resistance remains incomplete. This study investigated microRNA (miRNA) expression in olaparib-sensitive (PEO1, PEO4) and previously established olaparib-resistant OC cell lines (PEO1-OR) using high-throughput RT-qPCR and bioinformatic analyses. The role of miRNAs was explored regarding acquired resistance and resensitization with the ATR/CHK1 pathway inhibitors. Differentially expressed miRNAs were used to construct miRNA-mRNA regulatory networks and perform functional enrichment analyses for target genes with miRNet 2.0. TCGA-OV dataset was analyzed to explore the prognostic value of selected miRNAs and target genes in clinical samples. We identified potential processes associated with olaparib resistance, including cell proliferation, migration, cell cycle, and growth factor signaling. Resensitized PEO1-OR cells were enriched in growth factor signaling via PDGF, EGFR, FGFR1, VEGFR2, and TGFßR, regulation of the cell cycle via the G2/M checkpoint, and caspase-mediated apoptosis. Antibody microarray analysis confirmed dysregulated growth factor expression. The addition of the ATR/CHK1 pathway inhibitors to olaparib downregulated FGF4, FGF6, NT-4, PLGF, and TGFß1 exclusively in PEO1-OR cells. Survival and differential expression analyses for serous OC patients revealed prognostic miRNAs likely associated with olaparib resistance (miR-99b-5p, miR-424-3p, and miR-505-5p) and resensitization to olaparib (miR-324-5p and miR-424-3p). Essential miRNA-mRNA interactions were reconstructed based on prognostic miRNAs and target genes. In conclusion, our data highlight distinct miRNA profiles in olaparib-sensitive and olaparib-resistant cells, offering molecular insights into overcoming resistance with the ATR/CHK1 inhibitors in OC. Moreover, some miRNAs might serve as potential predictive signature molecules of resistance and therapeutic response.

Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression.

BACKGROUND: DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS: To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS: Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Predictive DNA damage signaling for low­dose ionizing radiation.

Numerous studies have attempted to develop biological markers for the response to radiation for broad and straightforward application in the field of radiation. Based on a public database, the present study selected several molecules involved in the DNA damage repair response, cell cycle regulation and cytokine signaling as promising candidates for low­dose radiation­sensitive markers. The HuT 78 and IM­9 cell lines were irradiated in a concentration­dependent manner, and the expression of these molecules was analyzed using western blot analysis. Notably, the activation of ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53 and H2A histone family member X (H2AX) significantly increased in a concentration­dependent manner, which was also observed in human peripheral blood mononuclear cells. To determine the radioprotective effects of cinobufagin, as an ATM and CHK2 activator, an in vivo model was employed using sub­lethal and lethal doses in irradiated mice. Treatment with cinobufagin increased the number of bone marrow cells in sub­lethal irradiated mice, and slightly elongated the survival of lethally irradiated mice, although the difference was not statistically significant. Therefore, KU60019, BML­277, pifithrin­α, and nutlin­3a were evaluated for their ability to modulate radiation­induced cell death. The use of BML­277 led to a decrease in radiation­induced p­CHK2 and γH2AX levels and mitigated radiation­induced apoptosis. On the whole, the present study provides a novel approach for developing drug candidates based on the profiling of biological radiation­sensitive markers. These markers hold promise for predicting radiation exposure and assessing the associated human risk.

Blocking the ATR-SerRS-VEGFA pathway targets angiogenesis for UV-induced cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, with an escalating incidence rate and a notable potential (up to 5%) for metastasis. Ultraviolet radiation (UVA and UVB) exposure is the primary risk factor for cSCC carcinogenesis, with literature suggesting ultraviolet radiation (UVR) promotes vascular endothelial growth factor A (VEGFA) expression. This study aims to investigate UVR-induced upregulation of VEGFA and explore combination therapeutic strategies. The skin squamous cell carcinoma cell line A431 was exposed to specific durations of ultraviolet radiation. The effect of emodin on ATR/SerRS/VEGFA pathway was observed. The cell masses were also transplanted subcutaneously into mice (n = 8). ATR inhibitor combined with emodin was used to observe the growth and angiogenesis of the xenografts. The results showed that UV treatment significantly enhanced the phosphorylation of SerRS and the expression level of VEGFA in A431 cells (p < 0.05). Treatment with emodin significantly inhibited this expression (p < 0.05), and the combination of emodin and ATR inhibitor further enhanced the inhibitory effect (p < 0.05). This phenomenon was further confirmed in the xenograft model, which showed that the combination of ATR inhibitor and emodin significantly inhibited the expression of VEGFA to inhibit angiogenesis (p < 0.05), thus showing an inhibitory effect on cSCC. This study innovatively reveals the molecular mechanism of UV-induced angiogenesis in cSCC and confirms SerRS as a novel target to inhibit cSCC angiogenesis and progression in vitro and in vivo studies.

Identification of ATM Mutation as a Potential Prognostic Biomarker for Immune Checkpoint Inhibitors Therapy.

BACKGROUND: Ataxia telangiectasia mutated (ATM), an apical DNA damage response gene, is a commonly mutated gene in tumors, and its mutation could strengthen tumor immunogenicity and alter the expression of PD-L1, which potentially contributes to immune checkpoint inhibitors (ICIs) therapy. METHODS: The characteristics of ATM mutation and its relationship with the ICIs-treated clinical prognosis have been analyzed comprehensively in this paper. The overall frequency of ATM mutations has been found to be 4% (554/10953) in the cancer genome atlas (TCGA) cohort. RESULTS: Both the TMB and MSI levels in patients with ATM mutations were significantly higher than those in patients without mutations (P < 0.0001). The median TMB was positively correlated with the frequency of ATM mutations (r = 0.54, P = 0.003). In the TCGA cohort, patients with ATM mutations had better clinical benefits in terms of overall survival (OS, hazard ratio (HR) = 0.736, 95% CI = 0.623 - 0.869), progression-free survival (PFS, HR = 0.761, 95% CI = 0.652 - 0.889), and disease-free survival (DFS, HR = 0.686, 95% CI = 0.512 - 0.919)] than patients without ATM mutations. Subsequently, the verification results showed ATM mutations to be significantly correlated with longer OS in ICIs-treated patients (HR = 0.710, 95% CI = 0.544 - 0.928). Further exploration indicated ATM mutation to be significantly associated with regulated anti-tumor immunity (P < 0.05). CONCLUSION: Our findings highlight the value of ATM mutation as a promising biomarker to predict ICIs therapy in multiple tumors.

Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours.

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

Bayesian meta-analysis of penetrance for cancer risk.

Multi-gene panel testing allows many cancer susceptibility genes to be tested quickly at a lower cost making such testing accessible to a broader population. Thus, more patients carrying pathogenic germline mutations in various cancer-susceptibility genes are being identified. This creates a great opportunity, as well as an urgent need, to counsel these patients about appropriate risk-reducing management strategies. Counseling hinges on accurate estimates of age-specific risks of developing various cancers associated with mutations in a specific gene, ie, penetrance estimation. We propose a meta-analysis approach based on a Bayesian hierarchical random-effects model to obtain penetrance estimates by integrating studies reporting different types of risk measures (eg, penetrance, relative risk, odds ratio) while accounting for the associated uncertainties. After estimating posterior distributions of the parameters via a Markov chain Monte Carlo algorithm, we estimate penetrance and credible intervals. We investigate the proposed method and compare with an existing approach via simulations based on studies reporting risks for two moderate-risk breast cancer susceptibility genes, ATM and PALB2. Our proposed method is far superior in terms of coverage probability of credible intervals and mean square error of estimates. Finally, we apply our method to estimate the penetrance of breast cancer among carriers of pathogenic mutations in the ATM gene.

Diverse pro-inflammatory ability of mutated spike protein derived from variant strains of SARS-CoV-2.

The severity of COVID-19 has been reported to differ among SARS-CoV-2 mutant variants. The overactivation of macrophages is involved in severe COVID-19, yet the effects of SARS-CoV-2 mutations on macrophages remain poorly understood. To clarify the effects, we examined whether mutations of spike proteins (S-proteins) affect macrophage activation. CD14+ monocyte-derived macrophages were stimulated with the recombinant S-protein of the wild-type, Delta, and Omicron strains or live viral particles of individual strains. Regarding IL-6 and TNF-α, Delta or Omicron S-protein had stronger or weaker pro­inflammatory ability, respectively, than the wild-type. Similar trends were observed between S-proteins and viral particles. S-protein mutations could be related to the diversity in macrophage activation and severity rates in COVID-19 caused by various SARS-CoV-2 strains.

Context matters: DNA virus infection reshapes DNA damage response pathways.

The cellular DNA damage response pathway can have vastly different outcomes depending on the source of its activation. Justice and colleagues apply phosphoproteomics to uncover a divergence in DNA-PK and ATM kinase activities in the contexts of DNA damage and DNA virus infection.

Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates.

Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

The KRAS, ATR and CHEK1 expression levels in endometrial cancer are the risk factors predicting recurrence.

Endometrial cancer is the most prevalent gynecologic malignancy with a high risk of recurrence. Local recurrence occurs in 7-20% of patients with treated stage I cancer within 3 years after primary treatment. In this study, we found significantly elevated mRNA expression levels of the oncoprotein KRAS, along with two replicative stress markers, ATR and CHEK1, in samples of endometrial carcinomas of endometrium (ECE) from patients with relapse. In contrast, mRNA expression levels of the studied genes were low and uniform in samples from patients without relapse. Elevated levels of KRAS protein and the phosphorylated form of ATR/CHEK1 were distinguishing features of recurrent ECE. A strong positive correlation was found between elevated mRNA and protein levels of the studied molecules. Elevated KRAS protein levels are characteristic of poorly differentiated (G3) endometrial carcinomas with deep myometrial invasion in patients without recurrence. In contrast, in patients with recurrence, higher protein levels of KRAS, pATR and pCHEK1 were observed in samples of G1-2 endometrial carcinomas, with statistically significant differences confirmed for pATR. High pCHEK1 protein levels are associated with deep tumor invasion in the myometrium among patients with recurrence. ROC analysis confirmed that evaluating the specificity and sensitivity of KRAS, pATR and pCHEK1 predicts recurrence development in patients with ECE. Our findings indicate that markers of replicative stress may play a significant role in ECE pathogenesis. Determining their levels in tumor samples after primary treatment could help define patients at high risk of recurrence and guide consequent courses of treatment.

Immunohistochemical findings and clinicopathological features of breast cancers with pathogenic germline mutations in Non-BRCA genes.

Deleterious germline mutations in multiple genes confer an increased breast cancer (BC) risk. Immunohistochemical (IHC) expression of protein products of mutated high-risk genes has not been investigated in BC. We hypothesized that pathogenic mutations may lead to an abnormal IHC expression pattern in the tumor cells. BCs with deleterious germline mutations in CHEK2, ATM, PALB2 & PTEN were identified. Immunohistochemistry was performed using Dako staining platform on formalin fixed paraffin embedded tumor tissue. Primary antibodies for PALB2 (ab202970), ATM [2C1(1A10)}, CHK2 (EPR4325), and PTEN (138G6) proteins were used for BCs with respective deleterious mutations. IHC expression was assessed in tumor and adjacent benign breast tissue. Total 27 BCs with 10 CHEK2, 9 ATM, 6 PALB2 & 2 PTEN deleterious germline mutations were identified. IHC staining was performed on 8 CHEK2, 7 ATM, 6 PALB2 & 2 PTEN cases. Abnormal CHEK2 IHC staining was identified in 7/8(88%) BCs. Three distinct CHK2 IHC patterns were noted: 1) Strong diffuse nuclear positivity (5 BC), 2) Null-pattern (2 BC), & 3) Normal breast-like staining in 1 BC Four of 5 (80%) strong CHK2 staining BC had missense CHEK2 mutations. Null-pattern was present with a missense & a frameshift mutation. Normal breast-like CHEK2 IHC staining pattern was present in 1 BC with CHEK2 frameshift mutation. Loss of nuclear/cytoplasmic PTEN IHC expression was noted in 2 in-situ carcinomas. Abnormal PTEN and CHK2 IHC were present in atypical ductal hyperplasia and flat epithelial atypia. ATM and PALB2 IHC expression patterns were similar in tumor cells and benign breast epithelium: mild to moderate intensity nuclear and cytoplasmic staining. We report abnormal CHEK2 IHC expression in 88% of BCs with pathogenic CHEK2 mutations. With PTEN and CHEK2 pathogenic mutations, abnormal IHC patterns are seen in early atypical proliferative lesions. IHC may be applied to identify CHEK2 & PTEN mutated BCs and precursor lesions.

Pyrroloquinoline quinone promotes human mesenchymal stem cell-derived mitochondria to improve premature ovarian insufficiency in mice through the SIRT1/ATM/p53 pathway.

BACKGROUND: DNA damage and oxidative stress induced by chemotherapy are important factors in the onset of premature ovarian insufficiency (POI). Studies have shown that mitochondria derived from mesenchymal stem cells (MSC-Mito) are beneficial for age-related diseases, but their efficacy alone is limited. Pyrroloquinoline quinone (PQQ) is a potent antioxidant with significant antiaging and fertility enhancement effects. This study aimed to investigate the therapeutic effect of MSC-Mito in combination with PQQ on POI and the underlying mechanisms involved. METHODS: A POI animal model was established in C57BL/6J mice by cyclophosphamide and busulfan. The effects of MSC-Mito and PQQ administration on the estrous cycle, ovarian pathological damage, sex hormone secretion, and oxidative stress in mice were evaluated using methods such as vaginal smears and ELISAs. Western blotting and immunohistochemistry were used to assess the expression of SIRT1, PGC-1α, and ATM/p53 pathway proteins in ovarian tissues. A cell model was constructed using KGN cells treated with phosphoramide mustard to investigate DNA damage and apoptosis through comet assays and flow cytometry. SIRT1 siRNA was transfected into KGN cells to further explore the role of the SIRT1/ATM/p53 pathway in combination therapy with MSC-Mito and PQQ for POI. RESULTS: The combined treatment of MSC-Mito and PQQ significantly restored ovarian function and antioxidant capacity in mice with POI. This treatment also reduced the loss of follicles at various stages, improving the disrupted estrous cycle. In vitro experiments demonstrated that PQQ facilitated the proliferation of MitoTracker-labelled MSC-Mito, synergistically restoring mitochondrial function and inhibiting oxidative stress in combination with MSC-Mito. Both in vivo and in vitro, the combination of MSC-Mito and PQQ increased mitochondrial biogenesis mediated by SIRT1 and PGC-1α while inhibiting the activation of ATM and p53, consequently reducing DNA damage-mediated cell apoptosis. Furthermore, pretreatment of KGN cells with SIRT1 siRNA reversed nearly all the aforementioned changes induced by the combined treatment. CONCLUSIONS: Our research findings indicate that PQQ facilitates MSC-Mito proliferation and, in combination with MSC-Mito, ameliorates chemotherapy-induced POI through the SIRT1/ATM/p53 signaling pathway.

Synergistic targeting of TrxR1 and ATM/AKT pathway in human colon cancer cells.

Thioredoxin reductase 1 (TrxR1) has emerged as a promising target for cancer therapy. In our previous research, we discovered several new TrxR1 inhibitors and found that they all have excellent anti-tumor activity. At the same time, we found these TrxR1 inhibitors all lead to an increase in AKT phosphorylation in cancer cells, but the detailed role of AKT phosphorylation in TrxR1 inhibitor-mediated cell death remains unclear. In this study, we identified the combination of AKT and TrxR1 inhibitor displayed a strong synergistic effect in colon cancer cells. Furthermore, we demonstrated that the synergistic effect of auranofin (TrxR1 inhibitor) and MK-2206 (AKT inhibitor) was caused by ROS accumulation. Importantly, we found that ATM inhibitor KU-55933 can block the increase of AKT phosphorylation caused by auranofin, and exhibited a synergistic effect with auranofin. Taken together, our study demonstrated that the activation of ATM/AKT pathway is a compensatory mechanism to cope with ROS accumulation induced by TrxR1 inhibitor, and synergistic targeting of TrxR1 and ATM/AKT pathway is a promising strategy for treating colon cancer.

Irradiated tumour cell-derived microparticles upregulate MHC-I expression in cancer cells via DNA double-strand break repair pathway.

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs.