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.

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.

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.

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.

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.

Association Between Polymorphisms in DNA Damage Repair Pathway Genes and Female Breast Cancer Risk.

Breast cancer risk have been discussed to be associated with polymorphisms in genes as well as abnormal DNA damage repair function. This study aims to assess the relationship between genes single nucleotide polymorphisms (SNPs) related to DNA damage repair and female breast cancer risk in Chinese population. A case-control study containing 400 patients and 400 healthy controls was conducted. Genotype was identified using the sequence MassARRAY method and expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) in tumor tissues was analyzed by immunohistochemistry assay. The results revealed that ATR rs13091637 decreased breast cancer risk influenced by ER, PR (CT/TT vs. CC: adjusted odds ratio [OR] = 1.54, 95% confidence interval [CI]: 1.04-2.27, p = 0.032; CT/TT vs. CC: adjusted OR = 1.63, 95%CI: 1.14-2.35, p = 0.008) expression. Stratified analysis revealed that PALB2 rs16940342 increased breast cancer risk in response to menstrual status (AG/GG vs. AA: adjusted OR = 1.72, 95%CI: 1.13-2.62, p = 0.011) and age of menarche (AG/GG vs. AA: adjusted OR = 1.54, 95%CI: 1.03-2.31, p = 0.037), whereas ATM rs611646 and Ku70 rs132793 were associated with reduced breast cancer risk influenced by menarche (GA/AA vs. GG: adjusted OR = 0.50, 95%CI: 0.30-0.95, p = 0.033). In a summary, PALB2 rs16940342, ATR rs13091637, ATM rs611646, and Ku70 rs132793 were associated with breast cancer risk.

Randomized Phase II Study of Gemcitabine With or Without ATR Inhibitor Berzosertib in Platinum-Resistant Ovarian Cancer: Final Overall Survival and Biomarker Analyses.

PURPOSE: The multicenter, open-label, randomized phase 2 NCI-9944 study (NCT02595892) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (hazard ratio [HR]=0.57, one-sided log-rank P = .044, which met the one-sided significance level of 0.1 used for sample size calculation). METHODS: We report here the final overall survival (OS) analysis and biomarker correlations (ATM expression by immunohistochemistry, mutational signature 3 and a genomic biomarker of replication stress) along with post-hoc exploratory analyses to adjust for crossover from gemcitabine to gemcitabine/berzosertib. RESULTS: At the data cutoff of January 27, 2023 (>30 months of additional follow-up from the primary analysis), median OS was 59.4 weeks with gemcitabine/berzosertib versus 43.0 weeks with gemcitabine alone (HR 0.79, 90% CI 0.52 to 1.2, one-sided log-rank P = .18). An OS benefit with addition of berzosertib to gemcitabine was suggested in patients stratified into the platinum-free interval ≤3 months (N = 26) subgroup (HR, 0.48, 90% CI 0.22 to 1.01, one-sided log-rank P =.04) and in patients with ATM-negative/low (N = 24) tumors (HR, 0.50, 90% CI 0.23 to 1.08, one-sided log-rank P = .06). CONCLUSION: The results of this follow-up analysis continue to support the promise of combined gemcitabine/ATRi therapy in platinum resistant ovarian cancer, an active area of investigation with several ongoing clinical trials.

DNA-PK and ATM drive phosphorylation signatures that antagonistically regulate cytokine responses to herpesvirus infection or DNA damage.

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-ß expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas.

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

Syk-dependent homologous recombination activation promotes cancer resistance to DNA targeted therapy.

Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase (PARP) inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase acknowledged for its regulatory roles in immune cell function, cell adhesion, and vascular development. This study presents evidence indicating that Syk expression in high-grade serous ovarian cancer and triple-negative breast cancers promotes DNA double-strand break resection, homologous recombination (HR), and subsequent therapeutic resistance. Our investigations reveal that Syk is activated by ATM following DNA damage and is recruited to DNA double-strand breaks by NBS1. Once localized to the break site, Syk phosphorylates CtIP, a pivotal mediator of resection and HR, at Thr-847 to promote repair activity, particularly in Syk-expressing cancer cells. Inhibition of Syk or its genetic deletion impedes CtIP Thr-847 phosphorylation and overcomes the resistant phenotype. Collectively, our findings suggest a model wherein Syk fosters therapeutic resistance by promoting DNA resection and HR through a hitherto uncharacterized ATM-Syk-CtIP pathway. Moreover, Syk emerges as a promising tumor-specific target to sensitize Syk-expressing tumors to PARP inhibitors, radiation and other DNA-targeted therapies.

ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability.

Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance.

Therapeutic Options Targeting the Ataxia-Telangiectasia Mutated (ATM)-mediated DNA Damage Response, Macropinocytosis, and Adaptive Immunity in Ovarian Cancer.

Ataxia-telangiectasia mutated (ATM) is a pivotal protein with versatile kinase activity that responds to DNA damage. While its well-established role as a DNA repair protein is widely recognized, the understanding of its noncanonical functions in ovarian cancer remains limited. Numerous studies have investigated the potential of targeting ATM for ovarian cancer treatment. In addition to its involvement in homologous recombination repair (HRR), an increasing body of research suggests that ATM plays a role in cellular metabolism and adaptive immunity. This review focuses on the current evidence and provides a perspective on how targeting ATM in ovarian cancer can address HRR-deficient genotypes, influence macropinocytosis, and enhance immune checkpoint blockade (ICB) therapy. It underscores the diverse avenues through which targeting ATM is a potential tailored treatment for ovarian cancer.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.