Protein Kinases ATR Research Articles

A phase I study of irinotecan combined with BAY1895344 (ATR inhibitor) in advanced solid tumors: Results of ETCTN 10402 dose escalation.

3077 Background: ATR protein kinase is activated by replication stress and recruited to stalled forks or single strand DNA defects in various cancers. The topoisomerase-I (Top1) inhibitor irinotecan induces DNA damage. The ATR inhibitor BAY1895344 (elimusertib) has demonstrated cytotoxic potential in SCLC and GI cancer xenografts when combined with Top1 inhibitors . Methods: This phase Ia trial assessed elimusertib in combination with irinotecan in adult patients with refractory advanced solid tumors for whom irinotecan can be considered standard care. Patients with previous irinotecan exposure were excluded. Two dosing schedules were used: 1) Biweekly - irinotecan IV (starting at 150 mg/m2) D1 + elimusertib (starting dose 20 mg PO BID) D1,D2 (cycle=14 days); 2) Weekly - irinotecan (starting at 50 mg/m2) IV on D1,8,15 with elimusertib (starting at 20 mg PO daily) on D2,D3,D9,D10 and D16,D17 (cycle=21 days). Dose escalation utilized a 3+3 design. Primary objectives were to assess safety and tolerability and estimate the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Secondary objectives included estimating pharmacokinetic (PK) profiles and assessing anti-tumor activity. Results: A total of 21 patients were enrolled in dose escalation (N=9 [Biweekly] and N=12 [Weekly], median age 58 and 2 lines of prior therapy. For the Biweekly cohort: 3/3 patients enrolled at dose level (DL) 1 experienced hematologic dose-limiting toxicity (DLT); 6 patients received DL-1 without any DLTs. The weekly regimen escalation comprised of 12 patients: 6 enrolled in DL1 with 2/6 having hematologic DLT and 5/6 unable to complete ≥75% of cycle 1 dosing; 6 enrolled in DL-1 with no DLTs observed. Notable grade 3+ treatment-related adverse events and summary best response are summarized (Table 1). The majority of evaluable patients in both schedules had initial disease control (56% [biweekly] and 67% [weekly]). One confirmed partial response was seen in the biweekly RP2D. Median progression-free survival was 2.1 mo [biweekly] and 2.5 mo [weekly]. PK results will be presented at time of meeting. Conclusions: Both RP2D and MTD of elimusertib in combination with irinotecan were reached for both dosing schedules: irinotecan 150 mg/m2 IV D1 + elimusertib 10 mg BID PO D1,D2 (biweekly) and irinotecan 25 mg/m2 IV on D1 + elimusertib (20 mg PO daily) on D2,D3 (weekly). Dose escalation was notably limited by myelotoxicity. Due to sponsor decision, the study was halted prior to planned expansion but the concept of ATR + topo I combination remains scientifically relevant. Clinical trial information: NCT04514497 . [Table: see text]

Protein kinase ATR inhibits E3 ubiquitin ligase CRL4PRL1 to stabilize ribonucleotide reductase in response to replication stress

The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4PRL1, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4PRL1 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.

Abstract 2405: Delineating molecular vulnerabilities of ATM mutant prostate cancers

Abstract Background: Mutations in DNA Damage Response (DDR) genes, including Ataxia, Telangiectasia, Mutated (ATM), are common in advanced castration-resistant prostate cancers (CRPCs). Poly (ADP-ribose) polymerase (PARP) inhibitors are approved in DDR mutant CRPCs, but demonstrate limited clinical efficacy in CRPCs with ATM mutations. In this project, we sought to specifically define the impact of ATM loss on DDR pathways in CRPC, with the goal of identifying alternate therapeutic vulnerabilities. Methods: ATM-KO CRPC cell lines were generated via CRISPR-Cas9 mediated knockout. ATM loss and abolishment of downstream ATM kinase activity was confirmed via western blot. We performed an unbiased phospho-proteomic evaluation of DDR pathways in parental and ATM-KO cells after ionizing radiation (IR). Clonogenic survival assays were performed with either an ATR inhibitor (VX970), the selective DNA-PKcs inhibitor (M3814), or combination therapy. Kinetics of DDR protein recruitment and resolution were interrogated with immunofluorescence (IF) staining for γH2ax, 53BP1, MDC1, and Rad51 foci. Results: ATM-KO cells were able to effectively repair DNA damage following IR, as measured by recruitment and resolution of γH2ax, 53BP1, MDC1, and Rad51 foci. Phospho-proteomic studies demonstrated that ATM-KO cells maintain canonical DDR pathways through ATR and DNA-PKcs kinase activation. Treatment of ATM-KO cells with either VX-970 or M3814 only incrementally affected DDR in ATM-KO cells compared to parental controls, as evidenced by clonogenic survival assays and maintenance of DDR foci. Importantly, combination treatment with VX-970 and M3814 prevented downstream DDR foci recruitment and radio-sensitized ATM-KO CRPC to a greater extent than parental controls. This suggested that activity of any of the trinity of kinases is sufficient to mediate DDR, and that blockade of both ATR and DNA-PKcs is required to effectively prevent DDR in ATM-KO CRPC. We then leveraged a RUVBL1 ATPase inhibitor Compound B, which significantly attenuates levels of these kinases in lung cancer cells. We confirmed that Compound B treatment attenuated ATR and DNA-PKcs protein expression and kinase activity in ATM-KO CRPC cells, and demonstrated sensitivity of ATM-KO cells to Compound B. Conclusions: Our data demonstrates that dual targeting of ATR and DNA-PKcs is necessary in ATM-KO CRPC, as either kinase is independently capable of mediating DDR following IR. Our initial studies indicate that the RUVBL1 ATPase inhibitor Compound B may effectively block DDR in ATM-mutant CRPC, and could be utilized as a novel therapeutic strategy in this molecular subtype. Citation Format: Mia Hofstad, Lan Yu, Andrea Woods, Zoi Sychev, Collin Gilbreath, Xiaofang Huo, Ralf Kittler, Justin M. Drake, Ganesh V. Raj. Delineating molecular vulnerabilities of ATM mutant prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2405.

1251P CHARIOT: A phase I dose escalation study combining ATR inhibitor Berzosertib with chemoradiotherapy in oesophageal cancer using time to event continual reassessment (TiTE-CRM) method: Results from A1 cohort (combination with palliative RT)

Berzosertib (M6220) is a selective ATR protein kinase inhibitor. Preclinical studies in esophageal cancer cell lines show that combining M6220 with cisplatin increased tumour cell kill in vitro and caused tumour growth delay in combination with radiation in vivo. The A1 cohort of CHARIOT (NCT03641547) tested the feasibility of combining Berzosertib with radiation in esophageal cancer. Primary objective: establish recommended phase II dose (RP2D) in combination with palliative RT; secondary objectives: safety, toxicity and efficacy.

P53 as a unique target of action of cisplatin in acute leukaemia cells.

Acute promyelocytic leukaemia (APL) occurs in approximately 10% of acute myeloid leukaemia patients. Arsenic trioxide (ATO) has been for APL chemotherapy, but recently several ATO‐resistant cases have been reported worldwide. Cisplatin (CDDP) enhances the toxicity of ATO in ovarian, lung cancer, chronic myelogenous leukaemia, and HL‐60 cells. Hence, the goal of this study was to investigate a novel target of CDDP action in APL cells, as an alternate option for the treatment of ATO‐resistant APL patients. We applied biochemical, molecular, confocal microscopy and advanced gene editing (CRISPR‐Cas9) techniques to elucidate the novel target of CDDP action and its functional mechanism in APL cells. Our main findings revealed that CDDP activated p53 in APL cells through stress signals catalysed by ATM and ATR protein kinases, CHK1 and CHK2 phosphorylation at Ser 345 and Thr68 residues, and downregulation and dissociation of MDM2‐DAXX‐HAUSP complex. Our functional studies confirmed that CDDP‐induced repression of MDM2‐DAXX‐HAUSP complex was significantly reversed in both nutilin‐3‐treated KG1a and p53‐knockdown NB4 cells. Our findings also showed that CDDP stimulated an increased number of promyelocytes with dense granules, activated p53 expression, and downregulated MDM2 in liver and bone marrow of APL mice. Principal conclusion of our study highlights a novel mode of action of CDDP targeting p53 expression which may provide a basis for designing new anti‐leukaemic compounds for treatment of APL patients.

Abstract 2588: M1774, a novel potent and selective ATR inhibitor, shows antitumor effects as monotherapy and in combination

Abstract The protein kinase ataxia telangiectasia- mutated and Rad3-related (ATR) is an important component of the DNA Damage Response (DDR) and a key mediator of the replication stress response (RSR). ATR is recruited to and activated by single-stranded DNA, which commonly forms as a consequence of replication stress (RS). ATR activation and signaling coordinates a multifaceted response to RS, however if left unresolved, replication forks may collapse, form double-strand breaks (DSB), lead to chromosomal rearrangements and eventually cell death. Several chemotherapeutic agents act by increasing RS in tumor cells, by directly damaging the DNA and/or depleting cellular resources required for DNA replication. Increased RS may also result from activation of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways even in the absence of exogenous DNA damaging insults. Inhibition of ATR activity in cancer cells disables the RSR, potentially enhancing the cytotoxicity of DNA damaging interventions and causing efficacy as monotherapy against tumors with reliance on ATR activity. M1774 is a potent inhibitor of ATR protein kinase with high selectivity towards other protein kinases. A broad range of antiproliferative activities (ranging from ~20 nM to >1 µM) was observed in a cancer cell line panel in vitro. M1774 showed pronounced synergy in combination with several DNA-damaging drugs and inhibitors of the DNA damage response. In vivo efficacy studies confirmed the combination potential suggested from in vitro studies. Pronounced combination benefit with complete tumor growth inhibition/regression was observed in combination with different chemotherapies or targeted DDR pathway inhibitors. Furthermore, M1774 showed pronounced activity as monotherapy in tumor models with gene mutations that result in a reliance on ATR activity. Clinical evaluation is ongoing to investigate M1774 as monotherapy and in combination with niraparib in patients with advanced solid tumors (DDRiver Solid Tumors 301, NCT04170153). Citation Format: Astrid Zimmermann, Heike Dahmen, Thomas Grombacher, Ulrich Pehl, Andree Blaukat, Frank T. Zenke. M1774, a novel potent and selective ATR inhibitor, shows antitumor effects as monotherapy and in combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2588.

Combination of the 6-thioguanine and disulfiram/Cu synergistically inhibits proliferation of triple-negative breast cancer cells by enhancing DNA damage and disrupting DNA damage checkpoint

Because of the lack of specific molecular targeted therapies, triple-negative breast cancer (TNBC) has high tumour recurrence and metastasis rates. It is urgent to develop novel chemotherapeutic strategies to improve patient survival. DNA damaging agents have been shown to sensitize cancer to genotoxic chemotherapies. We first found that 6-thioguanine (6-TG) can activate the NF-кB signalling pathway. Our results showed that NF-кB signalling was reduced when cells were treated with 6-TG/disulfiram (DSF)/Cu. DSF/Cu enhanced the 6-TG-mediated inhibition of proliferation. 6-TG/DSF/Cu inhibited cell cycle progression, causing cell cycle arrest in the S phase and G2/M phase. Moreover, the combined effect of 6-TG and DSF/Cu induced apoptosis, and either agent alone was able to induce apoptosis. The accumulation of γH2A indicated that DSF/Cu increased the DNA damage induced by 6-TG. Combined treatment with 6-TG and DSF/Cu synergistically reduced the levels of both phosphorylated and total ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), suggesting that DSF/Cu promoted 6-TG-induced DNA damage by suppressing ATR protein kinases, therefore enhancing cell apoptosis. In conclusion, we demonstrate that the combination of 6-TG and DSF/Cu exerted a significant synergistic antitumour effect on human TNBC in vitro and in vivo by enhancing DNA damage and disrupting DNA damage checkpoints. We propose that this combination therapy could be a novel strategy for the treatment of TNBC.

Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.

The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR–Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.

Structure-function analysis of TOPBP1\u2019s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system

The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.

Biochemical analysis of TOPBP1 oligomerization

TOPBP1 is an important scaffold protein that helps orchestrate the cellular response to DNA damage. Although it has been previously appreciated that TOPBP1 can form oligomers, how this occurs and the functional consequences for oligomerization were not yet known. Here, we use protein binding assays and other biochemical techniques to study how TOPBP1 self associates. TOPBP1 contains 9 copies of the BRCT domain, and we report that a subset of these BRCT domains interact with one another to drive oligomerization. An intact BRCT 2 domain is required for TOPBP1 oligomerization and we find that the BRCT1&2 region of TOPBP1 interacts with itself and with the BRCT4&5 pair. RAD9 and RHINO are two heterologous binding partners for TOPBP1's BRCT 1&2 domains, and we show that binding of these partners does not come at the expense of TOPBP1 oligomerization. Furthermore, we show that a TOPBP1 oligomer can simultaneously interact with both RAD9 and RHINO. Lastly, we find that the oligomeric state necessary for TOPBP1 to activate the ATR protein kinase is likely to be a tetramer.

Clinical and functional significance of tumor/stromal ATR expression in breast cancer patients

BackgroundMost breast cancer-associated fibroblasts (CAFs) are active and important cancer-promoting cells, with significant impact on patient prognosis. Therefore, we investigated here the role of the protein kinase ATR in breast stromal fibroblasts in the prognosis of locally advanced breast cancer patients.MethodsWe have used immunohistochemistry to assess the level of ATR in breast cancer tissues and their adjacent normal tissues. Immunoblotting as well as quantitative RT-PCR were utilized to show the role of breast cancer cells and IL-6 as well as AUF-1 in downregulating ATR in breast stromal fibroblasts. Engineered human breast tissue model was also used to show that ATR-deficient breast stromal fibroblasts enhance the growth of breast cancer cells.ResultsWe have shown that the protein kinase ATR is downregulated in cancer cells and their neighboring CAFs in breast cancer tissues as compared to their respective adjacent normal tissues. The implication of cancer cells in ATR knockdown in CAFs has been proven in vitro by showing that breast cancer cells downregulate ATR in breast fibroblasts in an IL-6/STAT3-dependent manner and via AUF-1. In another cohort of 103 tumors from locally advanced breast cancer patients, we have shown that absence or reduced ATR expression in tumoral cells and their adjacent stromal fibroblasts is correlated with poor overall survival as well as disease-free survival. Furthermore, ATR expression in CAFs was inversely correlated with tumor recurrence and progression.ConclusionATR downregulation in breast CAFs is frequent, procarcinogenic, and correlated with poor patient survival.

How do cells sense DNA lesions?

DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets. These targets, in turn, impact a wide range of cellular processes including DNA replication, DNA repair and cell cycle transitions. The importance of the DDR is highlighted by the fact that DDR inactivation is commonly found in cancer and causes many different human diseases. The protein kinases ATM and ATR, as well as their budding yeast orthologs Tel1 and Mec1, act as master regulators of the DDR. The initiating events in the DDR entail both DNA lesion recognition and assembly of protein complexes at the damaged DNA sites. Here, we review what is known about the early steps of the DDR.

ATR Kinase Activity Limits Mutagenesis and Promotes the Clonogenic Survival of Quiescent Human Keratinocytes Exposed to UVB Radiation

The ATR protein kinase has well-described roles in maintaining genomic integrity during the DNA synthesis phase of the cell cycle. However, ATR function in cells that are not actively replicating DNA remains largely unexplored. Using HaCaT and telomerase-immortalized human keratinocytes maintained in a confluent, nonreplicating state in vitro, ATR was found to be robustly activated in response to UVB radiation in a manner dependent on the nucleotide excision repair factor and DNA translocase XPB. Inhibition of ATR kinase activity under these conditions negatively impacted acute cell survival and cytotoxicity and severely inhibited the ability of UVB-irradiated HaCaT keratinocytes to proliferate upon stimulation with growth factors. Furthermore, ATR kinase inhibition in quiescent HaCaT keratinocytes potentiated UVB mutagenesis at the hypoxanthine phosphoribosyltransferase locus. Though ATR inhibition did not impact the rate of removal of cyclobutane pyrimidine dimers from genomic DNA, elevated levels of PCNA mono-ubiquitination and chromatin-associated PCNA and RPA indicate that excision gap-filling synthesis was altered in the absence of ATR signaling. These results indicate that the ATR kinase plays important roles in preventing mutagenesis and in promoting the proliferative potential of quiescent keratinocytes exposed to UVB radiation.

ATR kinase inhibition sensitizes quiescent human cells to the lethal effects of cisplatin but increases mutagenesis

The ATR protein kinase is known to protect cells from DNA damage induced during the replicative phase of the cell cycle. Small molecule ATR kinase inhibitors have therefore been developed to improve the effectiveness of DNA damage-based chemotherapy regimens aimed at killing rapidly proliferating tumor cells. However, whether ATR functions in a similar manner in non-replicating cells has not been examined and is important considering the fact that most cells in the body, including cancer stem cells in solid tumors, normally reside in either a quiescent or differentiated non-replicating state. Using cultured human cell lines maintained in a quiescent or slowly growing state in vitro, ATR was found to be activated following treatment with the common anti-cancer drug cisplatin in a manner dependent on the nucleotide excision repair (NER) system. Moreover, treatment with the ATR kinase inhibitors VE-821 and AZD6738 enhanced quiescent cell killing and apoptotic signaling induced by cisplatin. However, ATR kinase inhibition in quiescent cells treated with a low concentration of cisplatin also elevated the level of mutagenesis at the hypoxanthine phosphoribosyltransferase locus and resulted in increased levels of PCNA mono-ubiquitination. These results suggest that the excision gaps generated by NER may require a greater utilization of potentially mutagenic translesion synthesis polymerases in the absence of ATR kinase function. Thus, though ATR kinase inhibitors can aid in the killing of cisplatin-treated quiescent cells, such treatments may also result in a greater reliance on alternative mutagenic DNA polymerases to complete the repair of cisplatin-DNA adducts.

756 ATR kinase activity promotes the viability and proliferative capacity of quiescent human keratinocytes exposed to UVB radiation

The ATR protein kinase plays well-described roles during the DNA synthesis phase of the cell cycle in maintaining genomic integrity in response to DNA damage induced by UV radiation and other genotoxic agents. However, the function and activation mechanism of ATR in cells that are not actively replicating DNA remains largely unexplored but is physiologically important and relevant to understanding the fate of quiescent and stem cell populations exposed to DNA damaging agents. Using HaCaT and telomerase-immortalized normal human keratinocytes maintained in a confluent, non-replicating state in vitro, we show that the ATR kinase is rapidly activated in response to UVB radiation in a manner dependent on the DNA translocase XPB. Inhibition of ATR kinase activity has modest effects on acute cell survival but more severely abrogates the ability of UVB-irradiated keratinocytes to proliferate upon stimulation with growth factors. Furthermore, ATR kinase inhibition in quiescent HaCaT keratinocytes potentiates UVB mutagenesis at the HPRT locus but does not impact the rate of removal of cyclobutane pyrimidine dimers from genomic DNA. These results indicate that the ATR kinase plays important roles in limiting mutagenesis and promoting cell viability and proliferation following UVB exposure even in cells that are not actively synthesizing DNA.

ATR suppresses the pro-tumorigenic functions of breast stromal fibroblasts.

The ATR protein kinase is a master regulator of the cellular responses to DNA damage and replication stresses. Despite these crucial physiological roles, the implication of ATR in human carcinogenesis remains elusive. We have shown here that the ATR level is reduced in most cancer-associated fibroblasts (CAFs) as compared to their adjacent normal counterparts. Importantly, specific ATR knockdown activated breast fibroblasts, and enhanced their paracrine pro-carcinogenic effects via strong increase in the expression/secretion of SDF-1 and IL-6. Furthermore, ATR-deficient fibroblasts enhanced tumor growth and aggressiveness in orthotopic breast tumor xenografts. On the other hand, ectopic expression of ATR suppressed the expression/secretion of several cancer-promoting proteins such as IL-6, TGF-β1 and SDF-1, and inhibited the migration and invasion capacities of breast myofibroblast cells. Furthermore, ATR up-regulation in active breast fibroblasts reduced their paracrine pro-migratory/-invasive effects on breast cancer cells. Interestingly, the cancer promoting effects of ATR-deficient cells were repressed by ectopic expression of the ATR effector p53. These results indicate that ATR is a major target of cancer cells in breast fibroblasts wherein this protein kinase represses both autocrine and paracrine pro-carcinogenic effects. This indicates that the ATR status in these cells could be of great prognostic/diagnostic values.

ATR is required to complete meiotic recombination in mice

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

Abstract CT118: PK-Biomarker-Safety modelling aids choice of recommended Phase II dose and schedule for AZD6738 (ATR inhibitor)

Abstract Introduction: AZD6738 is a potent inhibitor of the ATR protein kinase that is being tested in patients with solid malignancies. In Phase I dose escalation studies, AZD6738 was dosed as monotherapy (continuous schedule from 40 - 480 mg per day), or in combination (intermittent schedule from 80 - 320 mg per day) with olaparib, durvalumab, carboplatin, or radiation. In those studies, thrombocytopenia was the main dose limiting toxicity and changes in peripheral monocytes and proliferating T-cells were seen with AZD6738, with up to 80% decrease on-drug and return to baseline upon cessation of dosing. This effect on monocytes and proliferating T cells was not observed with either single agent olaparib or durvalumab. Based on these observations, we developed two PKPD models linking AZD6738 dose, AZD6738 plasma exposure, and differential blood cell counts to support dose and schedule selection. Methods and Results: The first model characterized the quantitative relationship between the plasma exposure of AZD6738 monotherapy, and the difference between monocyte decrease (on-target activity) and platelet decrease (on-target toxicity). This model was a sigmoid function and predicted biological activity starting at exposures of 2.5 ug/mL (aligned with the in vitro IC90 in LoVo cells, and obtained in ≥ 50% of patients with 80 mg per day) increasing in a concentration dependent manner to 10 ug/mL (obtained in almost all patients dosed at 480 mg per day), after which negligible monocyte response over platelet decrease was observed. Those data were consistent with the preclinical observations which showed tumour regression after at least 3-day concomitant treatment of olaparib and AZD6738 at exposure maintaining IC90 [1]. The second model was a PK-safety model describing the temporal relationship between circulating platelets and exposure of AZD6738 administered in combination with olaparib dosed at 300 mg twice daily continuously. This model allowed generation of various platelet/ time profiles in the most sensitive patient group (10% of the patients) for different AZD6738 doses and schedules. The simulations indicated that, for doses considered biologically relevant by the first model, a period of 21-days free of AZD6738 is needed to give full recovery of potential thrombocytopenia in a 28-day cycle. Conclusion: Using monocyte and proliferative T-cell decreases as biological activity indicators and platelet decrease as a safety indicator, we quantified the exposure/activity/safety relationship for AZD6738. The recommended Phase II dose of AZD6738 (in combination with olaparib) was then driven by maintaining maximally active exposure consistent with manageable safety. Reference: [1]. 2017 AACR Abstract 2494. Acknowledgements: T.A. Yap (The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, London, UK) and J.C. Soria (Institut Gustave Roussy, Paris, France) Citation Format: Alienor Berges, S. Y. Amy Cheung, Andrew J. Pierce, Emma Dean, Brunella Felicetti, Nathan Standifer, Simon Smith, James Yates, Alan Lau, Christine Stephens, Matthew Krebs, Kevin Harrington, Simon J. Hollingsworth. PK-Biomarker-Safety modelling aids choice of recommended Phase II dose and schedule for AZD6738 (ATR inhibitor) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT118.

ATR inhibition controls aggressive prostate tumors deficient in Y-linked histone demethylase KDM5D.

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.

Insight into the role of PIKK family members and NF-\u043aB in DNAdamage-induced senescence and senescence-associated secretory phenotype of colon cancer cells

Senescence of cancer cells is an important outcome of treatment of many cancer types. Cell senescence is a permanent cell cycle arrest induced by stress conditions, including DNA damage. DNA damage activates DNA damage response (DDR), which involves members of the phosphatidylinositol 3-kinase-related kinase (PIKK) superfamily: protein kinases ATM, ATR, and DNA-PKcs. The so-far collected data indicate that ATM, with its downstream targets CHK2, p53, and p21, is the key protein involved in DDR-dependent senescence. It was also documented that the so-called senescence-associated secretory phenotype-SASP relies on ATM/CHK2, and not on p53 signaling. Moreover, genotoxic agents used in cancer treatment can activate NF-κB, which also induces transcription of SASP genes. In this paper, we have studied the involvement of three PIKK family members in colon cancer cell senescence and connection between DNA-damage-induced senescence and NF-κB-regulated SASP in p53-proficient and p53-deficient colon cancer cells treated with doxorubicin. We showed that doxorubicin induced cell senescence in both p53+/+ and p53−/− HCT116 cells, proving that this process is p53-independent. Senescence was successfully abrogated by a PIKK inhibitor, caffeine, or by simultaneous silencing of three PIKKs by specific siRNAs. By silencing individual members of PIKK family and analyzing common markers of senescence, the level of p21 and SA-β-Gal activity, we came to the conclusion that ATR kinase is crucial for the onset of senescence as, in contrast to ATM and DNA-PKsc, it could not be fully substituted by other PIKKs. Moreover, we showed that in case of silencing the three PIKKs, there was no SASP reduction accompanying the decrease in the level of p21 and SA-β-Gal (Senescence-Associated-β-Galactosidase) activity; whereas knocking down the NF-κB component, p65, abrogated SASP, but did not affect other markers of senescence, proving that DNA damage regulated senescence independently and NF-κB evoked SASP.