ATR Kinase Inhibitor Research Articles

Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.

Primary treatment modality for glioblastoma (GBM) post-surgery is radiation therapy. Due to increased DNA damage repair capacity of resistant residual GBM cells, recurrence is inevitable in glioblastoma and unfortunately the recurrent tumours are resistant to the conventional therapy. Here we used our previously described in vitro radiation survival model generated from primary GBM patient samples and cell lines, which recapitulates the clinical scenario of therapy resistance and relapse. Using the parent and recurrent GBM cells from these models, we show that similar to parent GBM, the recurrent GBM cells also elicit a competent DNA damage response (DDR) post irradiation. However, the use of apical DNA damage repair sensory kinase (ATM and/or ATR) is different in the recurrent cells compared to parent cells. Consistently, we demonstrate that there is a differential clonogenic response of parent and recurrent GBM cells to the ATM and ATR kinase inhibitors with recurrent samples switching between these sensory kinases for survival emphasizing on the underlying heterogeneity within and across GBM samples. Taken together, here we report that recurrent tumours utilize an alternate DDR kinase to overcome radiation induced DNA damage. Since there is no effective treatment specifically for recurred GBM patients, these findings provide a rationale for developing newer treatment option to sensitize recurrent GBM samples by detecting in clinics the ability of cells to activate a DNA damage repair kinase different from their parent counterparts.

Abstract P054: RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key mediator of cellular DNA damage repair (DDR) and is activated in response to DNA replication stress. ATR is attractive as a drug target in tumors with loss-of-function alterations in complimentary DDR pathways, including ataxia-telangiectasia mutated (ATM) and BRCA. RESULTS: RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor. RP-3500 is highly potent with ATR kinase IC50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. It is highly selective with >30-fold selectivity over mTOR and >2000-fold selectivity over ATM, DNA-PK and PI3Kα kinases. Preclinical tumor xenograft models harboring synthetic lethal (SNIPRx) gene mutations whose loss of function sensitizes to RP-3500 were selected for in vivo studies. RP-3500 treatment resulted in potent single-agent in vivo efficacy and/or tumor regression in multiple models at minimum effective doses (MED) of 5–7 mg/kg once daily. Pharmacodynamic assessments validated target engagement, with a proportional relationship between tumor pCHK1(Ser345) inhibition and circulating RP-3500 plasma levels (IC80 = 18.6 nM). Circulating free plasma levels of RP-3500 at the MED indicate that exposure above the in vivo tumor pCHK1(Ser345) IC80 for 10–12 hours is sufficient for efficacy and dose proportional phosphorylation of DNA damage markers γ-H2AX, pDNA-PKcs and pKAP1. In vitro, tumor cells with ATM loss exhibited increased susceptibility to RP-3500 and a 3-day compound exposure was sufficient to generate a sustained DNA damage response compared to cells with functional ATM expression. In ATM-deficient mouse models, short-duration intermittent (weekly 3 days on/4 days off or 5 days on/2 days off) dosing schedules maximized tumor growth inhibition while minimizing the impact on hematology parameters, including red blood cell depletion. These results emphasize the reversible nature of erythroid toxicity with RP-3500 and the advantage of intermittent dosing schedules to alleviate anemia. The 3 days on/4 days off intermittent treatment schedule also substantially improved the efficacy and tolerability of RP-3500 and PARP inhibitor combinations compared to continuous treatment schedules. Intermittent treatments of RP-3500 given concomitantly with reduced doses of olaparib or niraparib demonstrated synergistic efficacy in Granta-519 (ATM mutn) and SUM149PT (BRCA1mutn) models with minimal hematological adverse effects and superior efficacy to sequential treatment. CONCLUSIONS: These results provide a strong preclinical rationale to support clinical investigation of the novel ATR inhibitor RP-3500 on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a means of maximizing clinical benefit. RP-3500 is currently evaluated in the ongoing phase 1 TRESR (Treatment Enabled by SNIPRx) study (NCT04497116). Citation Format: Anne Roulston, Michal Zimmerman, Robert Papp, Alex Skeldon, Charles Pellerin, Émilie Dumas-Bérube, Valerie Dumais, Stephane Dorich, Sara Fournier, Li Li, Marie-Ève Leclaire, Shou Yun Yin, Amandine Chefson, Hunain Alam, William Yang, Chloe Fugère-Desjardins, Sabrina Hammond, Kathryn Skorey, Amina Mulani, Victoria Rimkunas, Artur Veloso, Martine Hamel, Rino Stocco, Yael Mamane, Zuomei Li, Jordan Young, Mike Zinda, Cameron Black. RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P054.

Targeting the DNA Damage Response Pathway in MLL- rearranged B-Acute Lymphoblastic Leukemias

Infant B-acute lymphoblastic leukemias (B-ALL) that harbor MLL-AF4 rearrangements are associated with a poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL - r ) childhood ALL (EFS > 85%). Thus, effective therapies that ensure long-term durable responses are of extreme interest. Recently, the targeting of the DNA damage response pathway (DDR) has been shown to be cytotoxic to MLL-r acute myeloid leukemia (AML) cells with oncogene driven replicative stress. In order to determine if targeting of the DDR pathway would be efficacious in MLL-r B-ALLs, we tested a panel of human AML and ALL cell lines and found that all B-ALL cell lines (those with and without MLL-r ) were highly sensitive to ATR kinase inhibition. At 72 hours, IC50s for MLL- r B-ALL cell lines (RS4;11, SEMK2, Hb11;19) were 2-3 fold lower than MLL-AF9 AML cell lines (Molm13, THP-1, Nomo1). Inhibition of ATR kinase induced double stranded DNA damage (as assessed by the increase in phospho-γH2AX levels), apoptosis and loss of the G2-M cell cycle checkpoint in a dose dependent manner in all cells treated, with B-ALL cells exhibiting these effects more rapidly and at lower doses.In order to better understand the mechanism behind the increased sensitivity of MLL -r B-ALLs to ATR inhibition, we generated a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in MLL-Af4 murine model. Recipient mice injected with Mll-Af4/N-RasG12D pre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable, with accelerated latency to secondary and tertiary disease. Multiple sequencing studies have indicated that while MLL -r B-ALL have relatively few additional somatic mutations, activating mutants of RAS seem to co-occur at a frequency close to 50%. Moreover, among MLL -r infant leukemias, those harboring mutant RAS mutations have even worse overall prognosis, further emphasizing the utility of our model.We were able to detect increased basal levels of DNA damage and replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to a small molecule inhibitor of ATR, AZ20. In vitro and in vivo treatment with AZ20 resulted in increased leukemia cytotoxicity. However, tertiary B-ALL transplanted mice eventually succumbed to disease following prolonged AZ20 treatment in vivo . As our model includes an activating mutation in N-Ras , we hypothesized that cells would be sensitive to the combination with small molecule inhibitors of downstream effectors of Ras. The combination of AZ20 and MEK inhibitor GSK212 (Trametinib) increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivo combination treatment of not only our mouse model but also of patient derived xenografts, harboring both mutations, exhibited decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. These data suggest a potent combination therapy for the treatment of this subgroup of poor prognosis MLL-AF4 patients with RAS mutations.To try to gain some further mechanistic insight, we conducted gene set enrichment analysis of RNA-sequencing of primary Mll-Af4/N-RasG12D leukemias and normal pro-B cells. Our analyses revealed significant negative correlations with gene signatures involving the G2/M checkpoint and ATR activation in response to replication stress, suggesting derangements in DDR pathways. We validated these observations with quantitative RT-PCR of ATR pathway members in murine mouse models of MLL-r AML and B-ALL. Our studies suggest that the observed sensitivity to ATR inhibition correlates to the overall expression level of ATR and ATR pathway members. We observed similar patterns of sensitivity and expression in the human cell lines we tested, as well as in an analysis of published gene expression data of infant and pediatric B-ALL patients. Altogether, our data indicates the inhibition of ATR in cells with oncogene driven replicative stress and abrogated ATR and ATR pathway expression, as part of an effective combination therapeutic strategy in this subtype of acute leukemia. DisclosuresArmstrong:C4 Therapeutics: Consultancy; Imago: Consultancy; Epizyme: Consultancy; Cyteir: Consultancy.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

SummarySARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses.

Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1’s ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.

Characterization of isogenic ovarian cancer cell line models of acquired resistance to the clinical ATR inhibitor AZD6738

Objective: Clinical ATR kinase inhibitors (ATRi) are an emerging class of therapeutics being used to treat diverse solid tumor malignancies, including ovarian cancer. This study is focused on improving our understanding of ATRi resistance through characterizing isogenic ovarian cancer cell line models of acquired resistance to the clinical ATR inhibitor AZD6738.

Characterization of SPK 98, a Torin2 analog, as ATR and mTOR dual kinase inhibitor

A series of Torin2, a second-generation ATP-competitive inhibitor, analogues were biologically characterized to identify their potential for ATR and mTOR kinase inhibition. Compound SPK 98 was observed to inhibit ATR/mTOR kinase selectively over ATM kinase in HCT 116 cell line. In addition to that, SPK 98 on 30 min incubation with human, mice and rat liver microsomes showed improved properties with an increased half-life (a maximum T ½ of 157 min) and internal clearance in mouse as compared to Torin2. Further, SPK 98 was also noticed to indulge in inducing premature chromatin condensation as a result of ATR/mTOR kinase inhibition at 50 nM. In a nutshell, our work presents the identification and characterization of SPK 98, a small molecule inhibitor, which exhibits improved specific inhibition for ATR at a lower concentration than Torin2.

DSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors.

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating γH2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (~ × 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of γH2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to γH2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoï tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside γH2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, γH2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 µm2), more nanofoci per focus (22.7 versus 13.2) and comparable nanofoci densities (~ 60 nanofoci/µm2). Scattered γH2AX nanofoci were equally present (~ 3 nanofoci/µm2), suggesting an endogenous origin. BLEO-treated cells were challenged with specific inhibitors of canonical H2AX kinases, namely: KU-55933, VE-821 and NU-7026 for ATM, ATR and DNA-PK, respectively. Under treatment with pooled inhibitors, clustered nanofoci vanished from super-resolution images while scattered nanofoci decreased (~ 50%) in density. Residual scattered nanofoci could reflect, among other alternatives, H2AX phosphorylation mediated by VRK1, a recently described non-canonical H2AX kinase. In addition to H2AX findings, an analytical approach to quantify clusters of highly differing density from SMLM data is put forward.

Preclinical evaluation of SC0245, a small molecule inhibitor of ATR kinase.

e15642 Background: The ataxia telangiectasia and Rad3-related (ATR) kinase is a member of the phosphoinositide 3-kinase related kinase (PIKK) family. ATR plays an important role in maintaining genome integrity during DNA replication through the activation of Chk1, and regulation of the DNA damage response (DDR). Replication stress (RS) is a major source of genomic instability in cancer, and targeting the RS-response kinase ATR has emerged as a promising antitumor approach. The purpose of this study was to investigate the antitumor activity of SC0245, a small molecule inhibitor of ATR kinase, in preclinical models of ATM pathway or ARID1A deficient solid tumors. Methods: The kinase inhibiting activity of SC0245 was determined using the ATR/ATRIP(h) complex assays. The cellular anti-proliferative activity was evaluated with tumor cells which was ATM pathway or ARID1A deficient. The in vivo antitumor activity of SC0245 was evaluated in ATM pathway or ARID1A deficient cell-derived xenograft (CDX) mouse models of gastric cancer (SNU-601) and colorectal adenocarcinoma (LoVo). Results: SC0245 displayed potent kinase inhibiting activity for ATR/ATRIP complex with IC50 14 nM, and had outstanding selectivity in the 104 onco-kinase panels. SC0245 significantly inhibited cell proliferation in ATM pathway or ARID1A deficient LoVo cells with IC50 0.163 μM, SNU-601 cells with IC50 0.218 μM. SC0245 showed excellent pharmacokinetics (PK) features with oral bioavailability ( > 80%) in mouse, rat and dog. Moreover, in the SNU601 and LoVo CDX mouse models, SC0245 oral administration significantly inhibited tumor growth, with better efficacy than AZD6738. Conclusions: SC0245, a novel potent ATR kinase inhibitor, has marked antitumor efficacy in the ATM pathway or ARID1A deficient solid tumor animal models, and has outstanding PK properties. SC0245 represents a promising clinical candidate for treating solid cancers, such as gastric and colorectal cancers.

Drug Targeting of Genomic Instability in Multiple Myeloma.

Genomic instability can be observed at both chromosomal and chromatin levels. Instability at the macro level includes centrosome abnormalities (CA) resulting in numerical as well as structural chromosomal changes, whereas instability at the micro level is characterized by defects in DNA repair pathways resulting in microsatellite instability (MIN) or mutations. Genomic instability occurs during carcinogenesis without impairing survival and growth, though the precise mechanisms remain unclear. Solid tumors arising from most cells of epithelial origin are characterized by genomic instability which renders them resistant to chemotherapy and radiotherapy. This instability is also observed in 25% of myeloma patients and has been shown to be highly prognostic, independently of the international staging system (ISS). However, a biomarker of aberrant DNA repair and loss of heterozygosity (LOH), was only observed at a frequency of 5% in newly diagnosed patients. Several new molecules targeting the pathways involved in genomic instability are under development and some have already entered clinical trials. Poly(ADP-ribose) polymerase-1 (PARP) inhibitors have been FDA-approved for the treatment of breast cancer type 1 susceptibility protein (BRCA1)-mutated metastatic breast cancer, as well as ovarian and lung cancer. Topoisomerase inhibitors and epigenetic histone modification-targeting inhibitors, such as HDAC (Histone Deacetylase) inhibitors which are novel agents that can target genomic instability. Several of the small molecule inhibitors targeting chromosomal level instability such as PARP, Akt, Aurora kinase, cyclin dependent kinase or spindle kinase inhibitors have been tested in mouse models and early phase I/II trials. ATM, ATR kinase inhibitors and DNA helicase inhibitors are also promising novel agents. However, most of these drugs are not effective as single agents but appear to act synergistically with DNA damaging agents such as radiotherapy, platinum derivatives, immunomodulators, and proteasome inhibitors. In this review, new drugs targeting genomic instability and their mechanisms of action will be discussed.

ATR signalling mediates the prosurvival function of phospho-NPM against PIDDosome mediated cell death

We uncover a novel non-canonical function of ATR kinase in the control of PIDDosome activation, and show that under normal cellular conditions involving no replication stress, ATR kinase controls the phosphorylation of cellular NPM via pChk1 as well as the two phosphatases, PPM1D and PP1β. We show that pNPM triggers the dissociation of NPM from PIDD, preventing the cell from undergoing caspase 2 mediated cell death via PIDDosome, thereby acting as an endogenous negative regulator of PIDDosome activation. pChk1 interaction with NPM is abrogated following ATR kinase inhibition, leading to the drop in nucleoplasmic/chromatin pNPM level, inducing PIDD. Consistent with this mechanism, the phosphomimic mutants of Chk1 and NPM become refractory to ATR/pChk1 kinase inhibition by avoiding PIDDosome signalling.

The Novel ATR Inhibitor BAY 1895344 Is Efficacious as Monotherapy and Combined with DNA Damage-Inducing or Repair-Compromising Therapies in Preclinical Cancer Models.

The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage-inducing or DNA repair-compromising cancer therapies by improving their efficacy.

ATR is a MYB regulated gene and potential therapeutic target in adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a rare cancer that preferentially occurs in the head and neck, breast, as well as in other sites. It is an aggressive cancer with high rates of recurrence and distant metastasis. Patients with advanced disease are generally incurable due to the lack of effective systemic therapies. Activation of the master transcriptional regulator MYB is the genomic hallmark of ACC. MYB activation occurs through chromosomal translocation, copy number gain or enhancer hijacking, and is the key driving event in the pathogenesis of ACC. However, the functional consequences of alternative mechanisms of MYB activation are still uncertain. Here, we show that overexpression of MYB or MYB-NFIB fusions leads to transformation of human glandular epithelial cells in vitro and results in analogous cellular and molecular consequences. MYB and MYB-NFIB expression led to increased cell proliferation and upregulation of genes involved in cell cycle control, DNA replication, and DNA repair. Notably, we identified the DNA-damage sensor kinase ATR, as a MYB downstream therapeutic target that is overexpressed in primary ACCs and ACC patient-derived xenografts (PDXs). Treatment with the clinical ATR kinase inhibitor VX-970 induced apoptosis in MYB-positive ACC cells and growth inhibition in ACC PDXs. To our knowledge, ATR is the first example of an actionable target downstream of MYB that could be further exploited for therapeutic opportunities in ACC patients. Our findings may also have implications for other types of neoplasms with activation of the MYB oncogene.

RAD50 regulates mitotic progression independent of DNA repair functions.

The Mre11A/RAD50/NBN complex (MRN) is an essential regulator of the cellular damage response after DNA double-strand breaks (DSBs). More recent work has indicated that MRN may also impact on the duration of mitosis. We show here that RAD50-deficient fibroblasts exhibit a marked delay in mitotic progression that can be rescued by lentiviral transduction of RAD50. The delay was observed throughout all mitotic phases in live cell imaging using GFP-labeled H2B as a fluorescent marker. In complementation assays with RAD50 phosphorylation mutants, modifications at Ser635 had little effect on mitotic progression. By contrast with RAD50, fibroblast strains deficient in ATM or NBN did not show a significant slowing of mitotic progression. Ataxia-telangiectasia-like disorder (ATLD) fibroblasts with nuclease-deficient MRE11A (p.W210C) tended to show slower mitosis, though by far not as significant as RAD50-deficient cells. Inhibitor studies indicated that ATM kinase activity might not grossly impact on mitotic progression, while treatment with MRE11A inhibitor PFM39 modestly prolonged mitosis. Inhibition of ATR kinase significantly prolonged mitosis but this effect was mostly independent of RAD50 status. Taken together, our data unravel a mitotic role of RAD50 that can be separated from its known functions in DNA repair.

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.

An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

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.

Berzosertib. ATR kinase inhibitor, Treatment of solid tumors

Berzosertib. ATR kinase inhibitor, Treatment of solid tumors

Ceralasertib. ATR kinase inhibitor, Treatment of cancer

Ceralasertib. ATR kinase inhibitor, Treatment of cancer