ATR, CHK1 and WEE1 inhibitors cause homologous recombination repair deficiency to induce synthetic lethality with PARP inhibitors.

PURPOSE: PARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1. EXPERIMENTAL DESIGN: A panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM). RESULTS: Rucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib. CONCLUSIONS: Our data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence "induced synthetic lethality" with PARPi.

Targeting the DNA damage response for cancer therapy.

The DNA damage response (DDR) is an elegant system, coordinating DNA repair with cell cycle checkpoints, that evolved to protect living organisms from the otherwise fatal levels of DNA damage inflicted by endogenous and environmental sources. Since many agents used to treat cancer; radiotherapy and cytotoxic chemotherapy, work by damaging DNA the DDR represents a mechanism of resistance. The original rational for the development of drugs to inhibit the DDR was to overcome this mechanism of resistance but clinical studies using this approach have not led to improvements in the therapeutic index. A more exciting approach is to exploit cancer-specific defects in the DDR, that represent vulnerabilities in the tumour and an opportunity to selectively target the tumour. PARP inhibitors (PARPi) selectively kill homologous recombination repair defective (HRD, e.g. through BRCA mutation) cells. This approach has proven successful clinically and there are now six PARPi approved for cancer therapy. Drugs targeting other aspects of the DDR are under pre-clinical and clinical evaluation as monotherapy agents and in combination studies. For this promising approach to cancer therapy to be fully realised reliable biomarkers are needed to identify tumours with the exploitable defect for monotherapy applications. The possibility that some combinations may result in toxicity to normal tissues also needs to be considered. A brief overview of the DDR, the development of inhibitors targeting the DDR and the current clinical status of such drugs is described here.

Differences in Durability of PARP Inhibition by Clinically Approved PARP Inhibitors: Implications for Combinations and Scheduling.

Six PARP inhibitors (PARPi) are approved for cancer therapy as monotherapy agents in daily or twice-daily continuous dosing schedules to maintain the necessary continuous suppression of PARP activity. Continuous PARP inhibition is required for single-agent anticancer activity. To investigate if such intense schedules are necessary, we determined the durability of PARP inhibition up to 72 h after a 1 h pulse of 1 µM of five of the approved PARPi, rucaparib, olaparib, niraparib, talazoparib and pamiparib, in IGROV-1 and ES-2 (human ovarian cancer) cells. Rucaparib caused the most persistent inhibition of PARP activity when maintained at ≥75% at 72 h after drug withdrawal in both IGROV-1 and ES-2 cells, but inhibition was more rapidly lost with the other PARPi. PARPi are also under clinical investigation with ATR inhibitors, and thus, we evaluated the implications for scheduling with an ATR inhibitor (VE-821). Rucaparib enhanced VE-821 cytotoxicity in co-exposure, sequential and delayed (24 h drug-free) schedules in IGROV-1 and ES-2 cells. Olaparib and niraparib enhanced VE-821 cytotoxicity only in co-exposed cells and not in sequential exposures. These data have clinical implications for the scheduling of PARPi as a monotherapy and in combination with ATR inhibitors and other cytotoxic drugs.

Determining the Potential of DNA Damage Response (DDR) Inhibitors in Cervical Cancer Therapy.

Cisplatin-based chemo-radiotherapy (CRT) is the standard treatment for advanced cervical cancer (CC) but the response rate is poor (46-72%) and cisplatin is nephrotoxic. Therefore, better treatment of CC is urgently needed. We have directly compared, for the first time, the cytotoxicity of four DDR inhibitors (rucaparib/PARPi, VE-821/ATRi, PF-477736/CHK1i and MK-1775/WEE1i) as single agents, and in combination with cisplatin and radiotherapy (RT) in a panel of CC cells. All inhibitors alone caused concentration-dependent cytotoxicity. Low ATM and DNA-PKcs levels were associated with greater VE-821 cytotoxicity. Cisplatin induced ATR, CHK1 and WEE1 activity in all of the cell lines. Cisplatin only activated PARP in S-phase cells, but RT activated PARP in the entire population. Rucaparib was the most potent radiosensitiser and VE-821 was the most potent chemosensitiser. VE-821, PF-47736 and MK-1775 attenuated cisplatin-induced S-phase arrest but tended to increase G2 phase accumulation. In mice, cisplatin-induced acute kidney injury was associated with oxidative stress and PARP activation and was prevented by rucaparib. Therefore, while all inhibitors investigated may increase the efficacy of CRT, the greatest clinical potential of rucaparib may be in limiting kidney damage, which is dose-limiting.

The Role of ATR Inhibitors in Ovarian Cancer: Investigating Predictive Biomarkers of Response.

Ataxia telangiectasia and Rad-3 related kinase (ATR) signals DNA lesions and replication stress (RS) to the S and G2/M checkpoints and DNA repair pathways making it a promising target to exploit the dysregulated DNA damage response in cancer. ATR inhibitors (ATRi) are under clinical investigation as monotherapy and in combination with other anticancer agents. Molecular determinants of sensitivity to ATRi are common in ovarian cancer, suggesting the therapeutic potential of ATRi. We investigated the cytotoxicity of the ATRi, VE-821, in a panel of human ovarian cancer cell lines. High grade serous (HGS) cell lines were significantly more sensitive to VE-821 than non-HGS (p ≤ 0.0001) but previously identified determinants of sensitivity (TP53, ATM and BRCA1) were not predictive. Only low RAD51 (p = 0.041), TopBP1 (p = 0.026) and APOBEC3B (p = 0.015) protein expression were associated with increased VE-821 sensitivity. HGS cells had increased levels of RS (pRPASer4/8 and γH2AX nuclear immunofluorescence), and elevated RS predicted sensitivity to VE-821 independently of the cell line subtype. These data suggest that functional assessment of RS biomarkers may be a better predictive biomarker of ATRi response than any single aberrant gene in ovarian cancer and potentially other cancers.

The Importance of Awareness, Acceptance, and Alignment With the Self: A Framework for Understanding Self-Connection.

We provide a theoretical framework for what it means to be self-connected and propose that self-connection is an important potential contributor to a person's well-being. We define self-connection as consisting of three components: 1) an awareness of oneself, 2) an acceptance of oneself based on this awareness, and 3) an alignment of one's behavior with this awareness. First, we position the concept within the broader self literature and provide the empirical context for our proposed definition of self-connection. We next compare and contrast self-connection to related constructs, including mindfulness and authenticity. Following, we discuss some of the potential relationships between self-connection and various aspects of mental health and well-being. Finally, we provide initial recommendations for future research, including potential ways to promote self-connection. In all, we present this theory to provide researchers with a framework for understanding self-connection so that they can utilize this concept to better support the efforts of researchers and practitioners alike to increase individuals' well-being in various contexts.

Radiotherapy biobanking: current landscape, opportunities, challenges, and future aspirations.

Half of all cancer patients receive radiotherapy, which makes a substantial contribution to their long-term disease control/cure. There are significant inter-patient differences in response, both in terms of efficacy and toxicity (frequently delayed onset) which are difficult to predict. With the introduction of technological improvements (e.g. stereotactic body radiotherapy and proton therapy) and development of combination therapies (e.g. radiotherapy and immune checkpoint inhibition), predictive biomarkers are needed even more. Whilst genomic studies have contributed significantly to predictions of response to anticancer therapy, there is no doubt that more information can be gathered from patient tissue samples. Patients are willing to donate their tissues to biobanks and wish them to be used as widely as possible for high-quality research. We report here a survey of the current practices in the UK from several groups collecting material from patients in radiotherapy trials and have identified barriers to collecting and sharing data and samples. We believe the current situation represents a significant missed opportunity to improve the personalisation of radiotherapy. We propose a greater involvement of patients and/or their advocates, a standardisation of the patient information leaflet, consent form content and data set, with easy linkage to clinical data, which would facilitate widespread sample and data discovery and availability to other researchers. The greater sharing of data and samples, nationally and internationally, would facilitate robust multicentre studies and avoid duplication of effort.

Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

Genomic, Transcriptomic, and Functional Alterations in DNA Damage Response Pathways as Putative Biomarkers of Chemotherapy Response in Ovarian Cancer.

Defective DNA damage response (DDR) pathways are enabling characteristics of cancers that not only can be exploited to specifically target cancer cells but also can predict chemotherapy response. Defective Homologous Recombination Repair (HRR) function, e.g., due to BRCA1/2 loss, is a determinant of response to platinum agents and PARP inhibitors in ovarian cancers. Most chemotherapies function by either inducing DNA damage or impacting on its repair but are generally used in the clinic unselectively. The significance of HRR and other DDR pathways in determining response to several other chemotherapy drugs is not well understood. In this study, the genomic, transcriptomic and functional analysis of DDR pathways in a panel of 14 ovarian cancer cell lines identified that defects in DDR pathways could determine response to several chemotherapy drugs. Carboplatin, rucaparib, and topotecan sensitivity were associated with functional loss of HRR (validated in 10 patient-derived primary cultures) and mismatch repair. Two DDR gene expression clusters correlating with treatment response were identified, with PARP10 identified as a novel marker of platinum response, which was confirmed in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Reduced non-homologous end-joining function correlated with increased sensitivity to doxorubicin, while cells with high intrinsic oxidative stress showed sensitivity to gemcitabine. In this era of personalised medicine, molecular/functional characterisation of DDR pathways could guide chemotherapy choices in the clinic allowing specific targeting of ovarian cancers.

Integration of computer-aided automated analysis algorithms in the development and validation of immunohistochemistry biomarkers in ovarian cancer.

In an era when immunohistochemistry (IHC) is increasingly depended on for histological subtyping, and IHC-determined biomarker informing rapid treatment choices is on the horizon; reproducible, quantifiable techniques are required. This study aimed to compare automated IHC scoring to quantify 6 DNA damage response protein markers using a tissue microarray of 66 ovarian cancer samples. Accuracy of quantification was compared between manual H-score and computer-aided quantification using Aperio ImageScope with and without a tissue classification algorithm. High levels of interobserver variation was seen with manual scoring. With automated methods, inclusion of the tissue classifier mask resulted in greater accuracy within carcinomatous areas and an overall increase in H-score of a median of 11.5% (0%-18%). Without the classifier, the score was underestimated by a median of 10.5 (5.2-25.6). Automated methods are reliable and superior to manual scoring. Fixed algorithms offer the reproducibility needed for high-throughout clinical applications.

PARPs, PAR and NAD Metabolism and Their Inhibitors in Cancer.

The role of poly(ADP-ribose) polymerase-1 (PARP1) in DNA repair and as a potential target for anticancer therapy has been under investigation for more than 50 years [...].

Poly(ADP-ribose) polymerase inhibition: past, present and future.

The process of poly(ADP-ribosyl)ation and the major enzyme that catalyses this reaction, poly(ADP-ribose) polymerase 1 (PARP1), were discovered more than 50 years ago. Since then, advances in our understanding of the roles of PARP1 in cellular processes such as DNA repair, gene transcription and cell death have allowed the investigation of therapeutic PARP inhibition for a variety of diseases - particularly cancers in which defects in DNA repair pathways make tumour cells highly sensitive to the inhibition of PARP activity. Efforts to identify and evaluate potent PARP inhibitors have so far led to the regulatory approval of four PARP inhibitors for the treatment of several types of cancer, and PARP inhibitors have also shown therapeutic potential in treating non-oncological diseases. This Review provides a timeline of PARP biology and medicinal chemistry, summarizes the pathophysiological processes in which PARP plays a role and highlights key opportunities and challenges in the field, such as counteracting PARP inhibitor resistance during cancer therapy and repurposing PARP inhibitors for the treatment of non-oncological diseases.

Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting.

In order to be effective models to identify biomarkers of chemotherapy response, cancer cell lines require thorough characterization. In this study, we characterised the widely used high grade serous ovarian cancer (HGSOC) cell line NIH-OVCAR3 using bioinformatics, cytotoxicity assays and molecular/functional analyses of DNA damage response (DDR) pathways in comparison to an ovarian cancer cell line panel. Bioinformatic analysis confirmed the HGSOC-like features of NIH-OVCAR3, including low mutation frequency, TP53 loss and high copy number alteration frequency similar to 201 HGSOCs analysed (TCGA). Cytotoxicity assays were performed for the standard of care chemotherapy, carboplatin, and DDR targeting drugs: rucaparib (a PARP inhibitor) and VE-821 (an ATR inhibitor). Interestingly, NIH-OVCAR3 cells showed sensitivity to carboplatin and rucaparib which was explained by functional loss of homologous recombination repair (HRR) identified by plasmid re-joining assay, despite the ability to form RAD51 foci and absence of mutations in HRR genes. NIH-OVCAR3 cells also showed high non-homologous end joining activity, which may contribute to HRR loss and along with genomic amplification in ATR and TOPBP1, could explain the resistance to VE-821. In summary, NIH-OVCAR3 cells highlight the complexity of HGSOCs and that genomic or functional characterization alone might not be enough to predict/explain chemotherapy response.

DNA damage checkpoint kinases in cancer.

DNA damage response (DDR) pathway prevents high level endogenous and environmental DNA damage being replicated and passed on to the next generation of cells via an orchestrated and integrated network of cell cycle checkpoint signalling and DNA repair pathways. Depending on the type of damage, and where in the cell cycle it occurs different pathways are involved, with the ATM-CHK2-p53 pathway controlling the G1 checkpoint or ATR-CHK1-Wee1 pathway controlling the S and G2/M checkpoints. Loss of G1 checkpoint control is common in cancer through TP53, ATM mutations, Rb loss or cyclin E overexpression, providing a stronger rationale for targeting the S/G2 checkpoints. This review will focus on the ATM-CHK2-p53-p21 pathway and the ATR-CHK1-WEE1 pathway and ongoing efforts to target these pathways for patient benefit.

ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms.

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04-0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

Repositioning PARP inhibitors for SARS-CoV-2 infection(COVID-19); a new multi-pronged therapy for acute respiratory distress syndrome?

Clinically approved PARP inhibitors (PARPi) have a mild adverse effect profile and are well tolerated as continuous daily oral therapy. We review the evidence that justifies the repurposing of PARPi to block the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and combat the life-threatening sequelae of coronavirus disease 2019 (COVID-19) by several mechanisms. PARPi can effectively decrease IL-6, IL-1 and TNF-α levels (key interleukins in SARS-CoV-2-induced cytokine storm) and can alleviate subsequent lung fibrosis, as demonstrated in murine experiments and clinical trials. PARPi can tune macrophages towards a tolerogenic phenotype. PARPi may also counteract SARS-CoV-2-induced and inflammation-induced cell death and support cell survival. PARPi is effective in animal models of acute respiratory distress syndrome (ARDS), asthma and ventilator-induced lung injury. PARPi may potentiate the effectiveness of tocilizumab, anakinra, sarilumab, adalimumab, canakinumab or siltuximab therapy. The evidence suggests that PARPi would benefit COVID-19 patients and trials should be undertaken.

Exploring the Synergy between PARP and CHK1 Inhibition in Matched BRCA2 Mutant and Corrected Cells.

PARP inhibition results in the accumulation of DNA SSBs, causing replication stress (RS) and lesions that can only be resolved by homologous recombination repair (HRR). Defects in HRR, e.g., due to BRCA2 mutation, confer profound sensitivity to PARP inhibitor (PARPi) cytotoxicity. In response to RS, CHK1 is activated to signal to S and G2/M cell cycle checkpoints and also to HRR. To determine the relative contribution of these two functions of CHK1 to survival following PARPi exposure, we investigated the effects of rucaparib (a PARPi) and PF-477736 (a CHK1 inhibitor) alone and in combination in cells with mutated and corrected BRCA2. The BRCA2 mutated V-C8 cells were 1000× more sensitive to rucaparib cytotoxicity than their matched BRCA2 corrected V-C8.B2 cells, but no more sensitive to PF-477736 despite having seven-fold higher levels of RS. PF-477736 caused a five-fold enhancement of rucaparib cytotoxicity in the V-C8.B2 cells, but no enhancement in the V-C8 cells. This differential sensitivity was not due to a difference in PARP1 or CHK1 expression or activity. PF-477736 increased rucaparib-induced RS (γH2AX foci) and completely inhibited RAD51 focus formation, indicating a profound suppression of HRR. Our data suggested that inhibition of HRR was the main mechanism of sensitisation to rucaparib, compounded with an inhibition of cell cycle checkpoints by PF-477736.

Targeting the DNA Damage Response for the Treatment of High Risk Neuroblastoma.

Despite intensive multimodal therapy, the survival rate for high risk neuroblastoma (HR-NB) remains <50%. Most cases initially respond to treatment but almost half will subsequently relapse with aggressive treatment resistant disease. Novel treatments exploiting the molecular pathology of NB and/or overcoming resistance to current genotoxic therapies are needed before survival rates can significantly improve. DNA damage response (DDR) defects are frequently observed in HR-NB including allelic deletion and loss of function mutations in key DDR genes, oncogene induced replication stress and cell cycle checkpoint dysfunction. Exploiting defects in the DDR has been a successful treatment strategy in some adult cancers. Here we review the genetic features of HR-NB which lead to DDR defects and the emerging molecular targeting agents to exploit them.