HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis.

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.

PTEN Depletion Increases Radiosensitivity in Response to Ataxia Telangiectasia-Related-3 (ATR) Inhibition in Non-Small Cell Lung Cancer (NSCLC).

Radiotherapy (RT) treatment is an important strategy for the management of non-small cell lung cancer (NSCLC). Local recurrence amongst patients with late-stage NSCLC remains a challenge. The loss of PTEN has been associated with radio-resistance. This study aimed to examine the efficacy of RT combined with ataxia telangiectasia-mutated Rad3-related (ATR) inhibition using Ceralasertib in phosphatase and tensin homolog (PTEN)-depleted NSCLC cells and to assess early inflammatory responses indicative of radiation pneumonitis (RP) after combined-modality treatment. Small hairpin RNA (shRNA) transfections were used to generate H460 and A549 PTEN-depleted models. Ceralasertib was evaluated as a single agent and in combination with RT in vitro and in vivo. Histological staining was used to assess immune cell infiltration in pneumonitis-prone C3H/NeJ mice. Here, we report that the inhibition of ATR in combination with RT caused a significant reduction in PTEN-depleted NSCLC cells, with delayed DNA repair and reduced cell viability, as shown by an increase in cells in Sub G1. Combination treatment in vivo significantly inhibited H460 PTEN-depleted tumour growth in comparison to H460 non-targeting PTEN-expressing (NT) cell-line-derived xenografts (CDXs). Additionally, there was no significant increase in infiltrating macrophages or neutrophils except at 4 weeks, whereby combination treatment significantly increased macrophage levels relative to RT alone. Overall, our study demonstrates that ceralasertib and RT combined preferentially sensitises PTEN-depleted NSCLC models in vitro and in vivo, with no impact on early inflammatory response indicative of RP. These findings provide a rationale for evaluating ATR inhibition in combination with RT in NSCLC patients with PTEN mutations.

Effect of ATR Inhibition in RT Response of HPV-Negative and HPV-Positive Head and Neck Cancers.

Radiotherapy (RT) has a central role in head and neck squamous cell carcinoma (HNSCC) treatment. Targeted therapies modulating DNA damage response (DDR) and more specific cell cycle checkpoints can improve the radiotherapeutic response. Here, we assessed the influence of ataxia-telangiectasia mutated and Rad3-related (ATR) inhibition with the ATR inhibitor AZD6738 on RT response in both human papillomavirus (HPV)-negative and HPV-positive HNSCC. We found that ATR inhibition enhanced RT response in HPV-negative and HPV-positive cell lines independent of HPV status. The radiosensitizing effect of AZD6738 was correlated with checkpoint kinase 1 (CHK1)-mediated abrogation of G2/M-arrest. This resulted in the inhibition of RT-induced DNA repair and in an increase in the percentage of micronucleated cells. We validated the enhanced RT response in HPV-negative and HPV-positive xenograft models. These data demonstrate the potential use of ATR inhibition in combination with RT as a treatment option for both HPV-negative and HPV-positive HNSCC patients.

Sulfoximines as ATR inhibitors: Analogs of VE-821.

The ATM- and Rad3-related (ATR) kinases play a key role in DNA repair processes and thus ATR is an attractive target for cancer therapy. Here we designed and synthesized sulfilimidoyl- and sulfoximidoyl-substituted analogs of the sulfone VE-821, a reported ATR inhibitor. The properties of these analogs have been investigated by calculating physicochemical parameters and studying their potential to specifically inhibit ATR in cells. Prolonged inhibition of ATR by the analogs in a Burkitt lymphoma cell line resulted in enhanced DNA damage and a substantial amount of apoptosis. Together our findings suggest that the sulfilimidoyl- and sulfoximidoyl-substituted analogs are efficient ATR inhibitors.

Targeting ATR Kinase as a Strategy for Canine Lymphoma and Leukaemia Treatment.

Ataxia telangiectasia and Rad3-related (ATR) kinase is one of the main regulators of cell response to DNA damage and replication stress. Effectiveness of ATR targeting in human cancers has been confirmed in preclinical studies and ATR inhibitors are currently developed clinically in human oncology. In the presented study, we tested the anticancer efficacy of ATR inhibitor berzosertib in an in vitro model of canine haematopoietic cancers. Using MTT assay and flow cytometry, we assessed the cytotoxicity of berzosertib in four established canine lymphoma and leukaemia cell lines and compared it with its activity against noncancerous canine cells. Further, we estimated the level of apoptosis in berzosertib-treated cells via flow cytometry and assessed H2AX phosphorylation as a marker of DNA damage using western blot technique. In flow-cytometric analysis, we also evaluated potential synergism between berzosertib and chlorambucil and assessed the influence of berzosertib on cell cycle disturbances induced by the drug. The results demonstrated that berzosertib, even without additional DNA damaging agent, can be effective against canine lymphoma and leukaemia cells at concentrations that were harmless for noncancerous cells, although sensitivity of individual cancer cell lines varied greatly. Cell death occurred through caspase-dependent apoptosis via induction of DNA damage. Berzosertib also acted synergistically with chlorambucil, probably by preventing DNA damage repair as a consequence of S-phase arrest abrogation. In conclusion, ATR inhibition may provide a new therapeutic option for the treatment of canine lymphomas and leukaemias, but further studies are required to determine potential biomarkers of their susceptibility.

Synergistic Activity of DNA Damage Response Inhibitors in Combination with Radium-223 in Prostate Cancer.

Radium-223 (223Ra) and Lutetium-177-labelled-PSMA-617 (177Lu-PSMA) are currently the only radiopharmaceutical treatments to prolong survival for patients with metastatic-castration-resistant prostate cancer (mCRPC); however, mCRPC remains an aggressive disease. Recent clinical evidence suggests patients with mutations in DNA repair genes associated with homologous recombination have a greater clinical benefit from 223Ra. In this study, we aimed to determine the utility of combining DNA damage response (DDR) inhibitors to increase the therapeutic efficacy of X-rays, or 223Ra. Radiobiological responses were characterised by in vitro assessment of clonogenic survival, repair of double strand breaks, cell cycle distribution, and apoptosis via PARP-1 cleavage. Here, we show that DDR inhibitors increase the therapeutic efficacy of both radiation qualities examined, which is associated with greater levels of residual DNA damage. Co-treatment of ATM or PARP inhibition with 223Ra increased cell cycle arrest in the G2/M phase. In comparison, combined ATR inhibition and radiation qualities caused G2/M checkpoint abrogation. Additionally, greater levels of apoptosis were observed after the combination of DDR inhibitors with 223Ra. This study identified the ATR inhibitor as the most synergistic inhibitor for both radiation qualities, supporting further pre-clinical evaluation of DDR inhibitors in combination with 223Ra for the treatment of prostate cancer.

Germline ATM Mutations Detected by Somatic DNA Sequencing in Lethal Prostate Cancer.

Background: Germline mutations in the ataxia telangiectasia mutated (ATM) gene occur in 0.5-1% of the overall population and are associated with tumour predisposition. The clinical and pathological features of ATM-mutated prostate cancer (PC) are poorly defined but have been associated with lethal PC. Objective: To report on the clinical characteristics including family history and clinical outcomes of a cohort of patients with advanced metastatic castration-resistant PC (CRPC) who were found to have germline ATM mutations after mutation detection by initial tumour DNA sequencing. Design setting and participants: We acquired germline ATM mutation data by saliva next-generation sequencing from patients with ATM mutations in PC biopsies sequenced between January 2014 and January 2022. Demographics, family history, and clinical data were collected retrospectively. Outcome measurements and statistical analysis: Outcome endpoints were based on overall survival (OS) and time from diagnosis to CRPC. Data were analysed using R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria). Results and limitations: Overall, seven patients (n = 7/1217; 0.6%) had germline ATM mutations detected, with five of them having a family history of malignancies, including breast, prostate, pancreas, and gastric cancer; leukaemia; and lymphoma. Two patients had concomitant somatic mutations in tumour biopsies in genes other than ATM, while two patients were found to carry more than one ATM pathogenic mutation. Five tumours in germline ATM variant carriers had loss of ATM by immunohistochemistry. The median OS from diagnosis was 7.1 yr (range 2.9-14 yr) and the median OS from CRPC was 5.3 yr (range 2.2-7.3 yr). When comparing these data with PC patients sequenced by The Cancer Genome Atlas, we found that the spatial localisation of mutations was similar, with distribution of alterations occurring on similar positions in the ATM gene. Interestingly, these include a mutation within the FRAP-ATM-TRRAP (FAT) domain, suggesting that this represents a mutational hotspot for ATM. Conclusions: Germline ATM mutations are rare in patients with lethal PC but occur at mutational hotspots; further research is warranted to better characterise the family histories of these men and PC clinical course. Patient summary: In this report, we studied the clinical and pathological features of advanced prostate cancers associated with germline mutations in the ATM gene. We found that most patients had a strong family history of cancer and that this mutation might predict the course of these prostate cancers, as well as response to specific treatments.

An orally available cancer drug AZD6738 prevents type 1 diabetes.

Type 1 diabetes (T1D) affects three million Americans, with 80 new people diagnosed each day. T1D is currently uncurable and there is an urgent need to develop additional drug candidates to achieve the prevention of T1D. We propose AZD6738 (ATRi), an orally available drug currently in phases I and II of clinical trials for various cancers, as a novel candidate to prevent T1D. Based on previously reported findings of ATRi inducing cell death in rapidly proliferating T cells, we hypothesized that this drug would specifically affect self-antigen activated diabetogenic T cells. These cells, if left unchecked, could otherwise lead to the destruction of pancreatic ß cells, contributing to the development of T1D. This work demonstrates that increasing the duration of ATRi treatment provides extended protection against T1D onset. Remarkably, 5-week ATRi treatment prevented T1D in a robust adoptive transfer mouse model. Furthermore, the splenocytes of animals that received 5-week ATRi treatment did not transfer immune-mediated diabetes, while the splenocytes from control animal transferred the disease in 10 days. This work shows that ATRi prevents T1D by specifically inducing cell death in self-antigen activated, highly proliferative diabetogenic T cells through the induction of DNA damage, resulting in the inhibition of IFNγ production and proliferation. These findings support the consideration of repurposing ATRi for T1D prevention.

7-Azaindole, 2,7-diazaindole, and 1H-pyrazole as core structures for novel anticancer agents with potential chemosensitizing properties.

Chemoresistance of cancer cells is a hallmark of treatment failure and the poor patient prognosis. The mechanism of resistance is often connected to the overexpression of specific kinases involved in DNA damage response cascade. Contrary, selected kinase inhibition can augment cancer cell sensitization to conventional therapy, enabling more efficient treatment. Among those kinases, ataxia-telangiectasia and Rad3-related kinase (ATR), the major responder to replication stress, stands out as one of the most attractive targets. Inspired by clinical candidates targeting ATR, we designed and prepared a small, focused library of 40 novel compounds building on 7-azaindoles, 2,7-diazaindoles, and 1H-pyrazoles as core structures. All the compounds alone or combined with cisplatin (CDDP) were screened against a panel of nine cancer cell lines and one healthy cell line. Three highlighted compounds (3, 22, and 29) were selected for broad oncology panel screening containing 104 kinases. Only compound 29, the 2,7-diazaindole representative, showed ATR inhibitory efficacy with the IC50 around 10 µM. In contrast, the compound 22, 7-azaindole congener with the most pronounced cytotoxicity profile exceeding CDDP alone or in combination with CDDP, expressed the multi-kinase activity. Highlighted representatives, including compound 29, were also effective alone against primary glioblastoma. Overall, we showed that 7-azaindole, and 2,7-diazaindole scaffolds could be considered novel pharmacophores delivering anticancer activity.

Efficient DNA Repair Mitigates Replication Stress Resulting in Less Immunogenic Cytosolic DNA in Radioresistant Breast Cancer Stem Cells.

Cancer stem cells (CSCs) are a major cause of tumor therapy failure. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies have shown that functional DNA repair via homologous recombination (HR) prevents radiation-induced accumulation of DNA in the cytoplasm, thereby inhibiting the intracellular immune response. However, it is unclear whether CSCs can suppress radiation-induced cytoplasmic dsDNA formation. Here, we show that the increased radioresistance of ALDH1-positive breast cancer stem cells (BCSCs) in S phase is mediated by both enhanced DNA double-strand break repair and improved replication fork protection due to HR. Both HR-mediated processes lead to suppression of radiation-induced replication stress and consequently reduction of cytoplasmic dsDNA. The amount of cytoplasmic dsDNA correlated significantly with BCSC content (p=0.0002). This clearly indicates that HR-dependent avoidance of radiation-induced replication stress mediates radioresistance and contributes to its immune evasion. Consistent with this, enhancement of replication stress by inhibition of ataxia telangiectasia and RAD3 related (ATR) resulted in significant radiosensitization (SER37 increase 1.7-2.8 Gy, p<0.0001). Therefore, disruption of HR-mediated processes, particularly in replication, opens a CSC-specific radiosensitization option by enhancing their intracellular immune response.

The antiproliferative effects of ataxia-telangiectasia mutated and ATM- and Rad3-related inhibitions and their enhancements with the cytotoxicity of DNA damaging agents in cholangiocarcinoma cells.

OBJECTIVE: To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines. METHODS: Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis. KEY FINDINGS: The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells. CONCLUSIONS: Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.

Advanced Prostate Cancer with ATM Loss: PARP and ATR Inhibitors.

BACKGROUND: Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. OBJECTIVE: To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. DESIGN, SETTING, AND PARTICIPANTS: We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. RESULTS AND LIMITATIONS: Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. CONCLUSIONS: ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. PATIENT SUMMARY: Of aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR.

ARID1A Regulates Transcription and the Epigenetic Landscape via POLE and DMAP1 while ARID1A Deficiency or Pharmacological Inhibition Sensitizes Germ Cell Tumor Cells to ATR Inhibition.

Germ cell tumors (GCTs) are the most common solid malignancies found in young men. Although they generally have high cure rates, metastases, resistance to cisplatin-based therapy, and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In a previous study, we identified downregulation of the chromatin-remodeling SWI/SNF complex member ARID1A as a key event in the mode of action of the histone deacetylase inhibitor romidepsin. Additionally, the loss-of-function mutations re-sensitize different tumor types to various drugs, like EZH2-, PARP-, HDAC-, HSP90- or ATR-inhibitors. Thus, ARID1A presents as a promising target for synthetic lethality and combination therapy. In this study, we deciphered the molecular function of ARID1A and screened for the potential of two pharmacological ARID1A inhibitors as a new therapeutic strategy to treat GCTs. By CRISPR/Cas9, we generated ARID1A-deficient GCT cells and demonstrate by mass spectrometry that ARID1A is putatively involved in regulating transcription, DNA repair and the epigenetic landscape via DNA Polymerase POLE and the DNA methyltransferase 1-associated protein DMAP1. Additionally, ARID1A/ARID1A deficiency or pharmacological inhibition increased the efficacy of romidepsin and considerably sensitized GCT cells, including cisplatin-resistant subclones, towards ATR inhibition. Thus, targeting ARID1A in combination with romidepsin and ATR inhibitors presents as a new putative option to treat GCTs.

PATRIOT: A phase I study to assess the tolerability, safety and biological effects of a specific ataxia telangiectasia and Rad3-related (ATR) inhibitor (AZD6738) as a single agent and in combination with palliative radiation therapy in patients with solid tumours.

PATRIOT is a phase I study of the ATR inhibitor, AZD6738, as monotherapy, and in combination with palliative radiotherapy. Here, we describe the protocol for this study, which opened in 2014 and is currently recruiting and comprises dose escalation of both drug and radiotherapy, and expansion cohorts.

The effect of caffeine on cisplatin-induced apoptosis of lung cancer cells.

BACKGROUND: Cisplatin is an important DNA-damaging anticancer drug that has been used to treat many cancer types. However, the effectiveness of cisplatin treatment diminishes quickly as cancer cells develop resistance to the drug, which eventually results in treatment failure. Caffeine is an ingredient contained in many food sources. Caffeine can inhibit activities of both ATM and ATR, two important protein kinases involved in DNA damage-induced cell cycle arrest and apoptosis. The effect of caffeine on cisplatin-based cancer treatment is not well known. METHODS: Caspase-3 activation and cell growth inhibition assays were used to determine the effect of caffeine on cisplatin-induced apoptosis and cell growth in lung cancer cells. Real time PCR, immunoblotting, and flow cytometry assays were used determine a mechanism through which the presence of caffeine increased cisplatin-induced apoptosis of the lung cancer cells. RESULTS: Our caspase-3 activation studies demonstrated that the presence of caffeine increased the cisplatin-induced apoptosis in both HTB182 and CRL5985 lung cancer cells. Our cell growth inhibition studies indicated that the presence of caffeine caused a more increase for cisplatin-induced cell growth inhibition. The results obtained from our real time PCR and western blot studies revealed that the presence of caffeine increased cisplatin-induced expression of the PUMA pro-apoptotic protein in these lung cancer cells. The results of our protein phosphorylation studies indicated that the presence of caffeine caused a decrease in CHK1 phosphorylation at Ser(317)/Ser(345) but an increase in ATM phosphorylation at Ser(1981) in the lung cancer cells treated with cisplatin. In addition, our flow cytometry studies also revealed that the presence of caffeine caused an increase in G1 cell population but a decrease for cisplatin-induced cell cycle arrests at the S and the G2 checkpoints in HTB182 and CRL5985 cells respectively. CONCLUSION: Our results suggest that the presence of caffeine increases the cisplatin-induced lung cancer cell killings by inhibiting ATR but inducing ATM activation, resulting in an increase in expression of PUMA protein and an increase in apoptosis.

Selective targeting of the G2/M cell cycle checkpoint to improve the therapeutic index of radiotherapy.

Despite tremendous advances in radiotherapy techniques, allowing dose escalation to tumour tissues and sparing of organs at risk, cure rates from radiotherapy or chemoradiotherapy remain suboptimal for most cancers. In tandem with our growing understanding of tumour biology, we are beginning to appreciate that targeting the molecular response to radiation-induced DNA damage holds great promise for selective tumour radiosensitisation. In particular, approaches that inhibit cell cycle checkpoint controls offer a means of exploiting molecular differences between tumour and normal cells, thereby inducing so-called cancer-specific synthetic lethality. In this overview, we discuss cellular responses to radiation-induced damage and discuss the potential of using G2/M cell cycle checkpoint inhibitors as a means of enhancing tumour control rates.

Mechanisms of Caffeine-Induced Inhibition of UVB Carcinogenesis.

Sunlight-induced non-melanoma skin cancer is the most prevalent cancer in the United States with more than two million cases per year. Several studies have shown an inhibitory effect of caffeine administration on UVB-induced skin cancer in mice, and these studies are paralleled by epidemiology studies that indicate an inhibitory effect of coffee drinking on non-melanoma skin cancer in humans. Strikingly, decaffeinated coffee consumption had no such inhibitory effect. Mechanism studies indicate that caffeine has a sunscreen effect that inhibits UVB-induced formation of thymine dimers and sunburn lesions in the epidermis of mice. In addition, caffeine administration has a biological effect that enhances UVB-induced apoptosis thereby enhancing the elimination of damaged precancerous cells, and caffeine administration also enhances apoptosis in tumors. Caffeine administration enhances UVB-induced apoptosis by p53-dependent and p53-independent mechanisms. Exploration of the p53-independent effect indicated that caffeine administration enhanced UVB-induced apoptosis by inhibiting the UVB-induced increase in ATR-mediated formation of phospho-Chk1 (Ser345) and abolishing the UVB-induced decrease in cyclin B1 which resulted in caffeine-induced premature and lethal mitosis in mouse skin. In studies with cultured primary human keratinocytes, inhibition of ATR with siRNA against ATR inhibited Chk1 phosphorylation and enhanced UVB-induced apoptosis. Transgenic mice with decreased epidermal ATR function that were irradiated chronically with UVB had 69% fewer tumors at the end of the study compared with irradiated littermate controls with normal ATR function. These results, which indicate that genetic inhibition of ATR (like pharmacologic inhibition of ATR via caffeine) inhibits UVB-induced carcinogenesis support the concept that ATR-mediated phosphorylation of Chk1 is an important target for caffeine's inhibitory effect on UVB-induced carcinogenesis.