ATR Inhibitor Research Articles

Glioma Cell Invasion Is Dependent Upon the DNA Damage Response Kinase ATR

Abstract AIMS Gliomas have high levels of DNA replication stress and clinical trials of inhibitors of the key replication stress response protein ATR (ataxia telangiectasia and rad 3 related protein) as radiosensitisers are planned. We aimed to investigate the effect of ATR inhibition on the ability of glioma cells to infiltrate and invade the brain. METHOD Live cell imaging in a panel of primary glioma cultures following siRNA or pharmacological inhibition of ATR. Invasion following treatment of murine orthotopic gliomas was determined by immunohistochemistry. Intravital imaging of GFP expressing murine orthotopic xenografts via an intracranial window model of glioma was undertaken. RESULTS Invading margins of human glioma samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated reduced cell velocity following ATR inhibition (Berzosertib/BAY1895344) or siRNA. Cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures which engulf high molecular weight dextran, compatible with blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed sequestration of integrins within macropinosomes and reduced integrin cycling. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further in vivo study demonstrated a reduction in invading tumour cells. CONCLUSION We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin recycling in GBM cells which results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.

282 (PB062) - Optimization of treatment schedule for the ATR inhibitor elimusertib (BAY 1895344) in preclinical tumor models

Background: ATR kinase is a critical component of the DNA damage response (DDR) machinery. Elimusertib, a potent and selective ATR inhibitor, has demonstrated efficacy in preclinical tumor models as monotherapy and synergistic antitumor activity in combination with DNA damage inducing as well as DNA repair compromising therapies. Elimusertib monotherapy revealed clinical benefit in patients with advanced solid tumors carrying DDR defects (Yap et al. Cancer Discovery 2021; Yap et al. AACR Annual Meeting 2022).

279 (PB059) - ATR inhibitor camonsertib (RP-3500) suppresses early-stage erythroblasts by mediating ferroptosis

Background: Ataxia telangiectasia mutated and Rad3-related kinase (ATR) mediates cellular response to replication stress and DNA damage. Camonsertib is a potent and selective ATR inhibitor with strong pre-clinical efficacy, promising clinical activity (NCT04497116) but results in rapid monocytopenia leading to dose limiting anemia potentially implicating a disturbance in iron homeostasis. Pre-clinically, dosing 3 days on/4 off mitigates anemia without compromising efficacy, allowing reticulocyte regeneration between doses.

277 (PB057) - PPP2R1A missense mutations as a novel biomarker of response to ATR inhibitors in ARID1A mutant ovarian clear cell carcinoma

Introduction: Ovarian clear cell carcinoma (OCCC) is characterised by a high prevalence of ARID1A mutations and chemotherapy resistance. We previously found that loss of ARID1A causes ATR inhibitor (ATRi) sensitivity, a finding that led to the Phase 2 ATARI clinical trial (NCT04065269). We aim to identify novel genetic determinants of ATRi response in ARID1A-mutant OCCC.

19 (PB005) - DNA Damage Response (DDR) Basket of Baskets (D-BOB) Trial: Phase 1/2 Study of the ATR inhibitor (ATRi) berzosertib and PD-L1 inhibitor avelumab in patients (pts) with advanced solid tumors with DDR molecular alterations

Background: ATR is a central kinase involved in the DDR and replication stress response (RSR) and innate immune activation. This is the first report of the investigator-initiated phase 1/2 D-BOB trial (NCT04266912), funded by EMD Serono (CrossRef Funder ID: 10.13039/100004755).

278 (PB058) - CDK12 loss leads to replication stress and sensitivity to combinations of the ATR inhibitor camonsertib (RP-3500) with PARP inhibitors

Background: Loss-of-function (LOF) alterations in Cyclin-Dependent Kinase 12 (CDK12) are found across tumors, including metastatic prostate (∼5%) and serous ovarian (∼3%) cancers. While initial data suggested that CDK12 LOF compromises DNA repair by homologous recombination (HR), there is burgeoning evidence questioning this notion. CDK12-deficient tumors lack genomic features indicative of HR-deficiency (HRD) and instead harbor large tandem duplications thought to originate from DNA replication stress of unknown etiology.

Selective vulnerability of ARID1A deficient colon cancer cells to combined radiation and ATR-inhibitor therapy.

ARID1A is frequently mutated in colorectal cancer (CRC) cells. Loss of ARID1A function compromises DNA damage repair and increases the reliance of tumor cells on ATR-dependent DNA repair pathways. Here, we investigated the effect of ionizing radiation (IR), in combination with ATR inhibitors (ATRi) in CRC cell lines with proficient and deficient ARID1A. The concept of selective vulnerability of ARID1A deficient CRC cells to ATRi was further tested in an ex vivo system by using the ATP-tumor chemosensitivity assay (ATP-TCA) in cells from untreated CRC patients, with and without ARID1A expression. We found selective sensitization upon ATRi treatment as well as after combined treatment with IR (P<0.001), especially in ARID1A deficient CRC cells (P <0.01). Knock-down of ARID1B further increased the selective radiosensitivity effect of ATRi in ARID1A negative cells (P<0.01). Mechanistically, ATRi abrogates the G2 checkpoint (P<0.01) and homologous recombination repair (P<0.01) in ARID1A deficient cells. Most importantly, ex-vivo experiments showed that ATRi had the highest radiosensitizing effect in ARID1A negative cells from CRC patients. Collectively, our results generate pre-clinical and clinical mechanistic rationale for assessing ARID1A defects as a biomarker for ATR inhibitor response as a single agent, or in a synthetic lethal approach in combination with IR.

Multiple-low-dose therapy: effective killing of high-grade serous ovarian cancer cells with ATR and CHK1 inhibitors.

High-grade serous ovarian cancer (HGSOC) is an aggressive disease that typically develops drug resistance, thus novel biomarker-driven strategies are required. Targeted therapy focuses on synthetic lethality-pioneered by PARP inhibition of BRCA1/2-mutant disease. Subsequently, targeting the DNA replication stress response (RSR) is of clinical interest. However, further mechanistic insight is required for biomarker discovery, requiring sensitive models that closely recapitulate HGSOC. We describe an optimized proliferation assay that we use to screen 16 patient-derived ovarian cancer models (OCMs) for response to RSR inhibitors (CHK1i, WEE1i, ATRi, PARGi). Despite genomic heterogeneity characteristic of HGSOC, measurement of OCM proliferation was reproducible and reflected intrinsic tumour-cell properties. Surprisingly, RSR targeting drugs were not interchangeable, as sensitivity to the four inhibitors was not correlated. Therefore, to overcome RSR redundancy, we screened the OCMs with all two-, three- and four-drug combinations in a multiple-low-dose strategy. We found that low-dose CHK1i-ATRi had a potent anti-proliferative effect on 15 of the 16 OCMs, and was synergistic with potential to minimise treatment resistance and toxicity. Low-dose ATRi-CHK1i induced replication catastrophe followed by mitotic exit and post-mitotic arrest or death. Therefore, this study demonstrates the potential of the living biobank of OCMs as a drug discovery platform for HGSOC.

Targeting DNA damage response components induces enhanced STING-dependent type-I IFN response in ATM deficient cancer cells and drives dendritic cell activation

ABSTRACT The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM −/− FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.

457MO A phase I study of ATR inhibitor M1774 in patients with solid tumours (DDRiver Solid Tumours 301): Part A1 results

Ataxia telangiectasia and Rad3-related (ATR) protein kinase plays a critical role in the DNA damage response. M1774, a potent, selective, orally administered ATR inhibitor, exerts antitumour activity in preclinical models.

485P Correlation of clinical, genomic and hematological parameters with ATR inhibitor (ATRi) outcomes in phase I/II clinical trials

ATR inhibition is an emerging strategy in tumors harboring elevated replicative stress. We sought to identify clinical, genomic and hematological parameters associated with ATRi benefit, and to delineate potential links between ATRi toxicity with efficacy.

OA15.05 HUDSON: An Open-Label, Multi-Drug, Biomarker-Directed Phase 2 Study in NSCLC Patients Who Progressed on Anti-PD-(L)1 Therapy

HUDSON (NCT03334617) is a multi-arm umbrella trial for patients with advanced non-small-cell lung cancer (NSCLC) previously treated with platinum-doublet chemotherapy and anti-PD-(L)1 immunotherapy (separately or in combination) who had disease progression on prior anti-PD-(L)1 therapy. The study evaluates the efficacy, safety, and tolerability of multiple treatment combinations tailored by molecular alteration, with the goal of overcoming resistance to PD-(L)1 blockade. Here we present mature efficacy and safety results for the initial combinations - durvalumab plus: olaparib (PARP inhibitor; Module 1), danvatirsen (STAT3 inhibitor; Module 2), ceralasertib (ATR inhibitor; Module 3), and oleclumab (anti-CD73 antibody; Module 5).

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.

656MO Phase I study of M6620 (VX-970, berzosertib) in combination with cisplatin and XRT in patients with locally advanced head and neck squamous cell carcinoma

M6620 is a highly potent and selective ATP-competitive ATR inhibitor. It showed strong synergy with cisplatin and radiation in preclinical cancer models. The significant vulnerability to ATR inhibition by cancer cells harboring inactivating p53 mutations provided a rationale to test the safety of M6620 along with definitive chemoradiation in Head and Neck Squamous Cell Carcinoma (HNSCC) where such alterations are common.

484P CHARIOT trial (cohort A2): A phase I dose-escalation study combining the ATR inhibitor berzosertib with cisplatin and capecitabine

Berzosertib is a potent inhibitor of ataxia telangiectasia-mutated and Rad3-related protein kinase (ATR). Preclinical studies have shown that ATR inhibition enhances the cytotoxic effects of DNA damaging drugs. Cohort A2 of the CHARIOT trial (NCT03641547) explored the combination of berzosertib with cisplatin and capecitabine in advanced solid tumours to assess the optimal treatment schedule, safety and preliminary efficacy.

Immunogenicity of small-cell lung cancer associates with STING pathway activation and is enhanced by ATR and TOP1 inhibition.

The activation of STING (stimulator of interferon genes) pathway enhances antitumor immunity in small-cell lung cancer (SCLC), while the DNA damage induced by non-cGAMP-based agonists is a potent inducer of STING activity. Here, we investigate the intrinsic expression of STING in cancer cells and evaluate the value of the combination of ATR and TOP1 inhibitors in enhancing antitumor immunity. STING expression was assessed at mRNA and protein levels in SCLC and normal lung tissues. Transcriptomic subsets of SCLC were identified based on STING-related genes. Distinct mutation and immunogenomic profiles of these subsets were determined. The direct antitumor efficacy and the potential of enhancing antitumor immunity of the strategy using the ATR-TOP1-inhibitor combination were tested in SCLC cell lines. The intrinsic expression of STING was significantly reduced in SCLC compared to normal lung tissues (p < 0.0001). Three STING-related SCLC subtypes were identified in which the STING-high subtype was associated with (1) high immune infiltration, (2) high expression of genes related to MHC and immune checkpoints, and (3) high EMT and ferroptosis score. On the contrary, the STING-low subtype was enriched with pathways related to DNA damage response (DDR) and cell cycle progression. The association between the DDR pathway activity and the STING-IFN innate immune response was verified by in vitro experiments in which the inhibition of ATR and TOP1 triggered the expression of genes encoding type I IFN signaling and pro-inflammatory cytokines/chemokines in a STING-low SCLC cell line. Our study verifies that activation of the STING-IFN response by ATR and TOP1 inhibitors might be a therapeutic strategy to improve the response to immune checkpoint therapy in STING-low SCLC. Furthermore, the combinations of ATR and TOP1 inhibitors can augment tumor inflammation in STING-low SCLC.

Targeted Inhibition of DNA-PKcs, ATM, ATR, PARP, and Rad51 Modulate Response to X Rays and Protons.

Small molecule inhibitors are currently in preclinical and clinical development for the treatment of selected cancers, particularly those with existing genetic alterations in DNA repair and DNA damage response (DDR) pathways. Keen interest has also been expressed in combining such agents with other targeted antitumor strategies such as radiotherapy. Radiotherapy exerts its cytotoxic effects primarily through DNA damage-induced cell death; therefore, inhibiting DNA repair and the DDR should lead to additive and/or synergistic radiosensitizing effects. In this study we screened the response to X-ray or proton radiation in cell lines treated with DDR inhibitors (DDRis) targeting ATM, ATR, DNA-PKcs, Rad51, and PARP, with survival metrics established using clonogenic assays. We observed that DDRis generate significant radiosensitization in cancer and primary cells derived from normal tissue. Existing genetic defects in cancer cells appear to be an important consideration when determining the optimal inhibitor to use for synergistic combination with radiation. We also show that while greater radiosensitization can be achieved with protons (9.9 keV/µm) combined with DDRis, the relative biological effectiveness is unchanged or in some cases reduced. Our results indicate that while targeting the DDR can significantly radiosensitize cancer cells to such combinations, normal cells may also be equally or more severely affected, depending on the DDRi used. These data highlight the importance of identifying genetic defects as predictive biomarkers of response for combination treatment.

Quantitation of the ataxia-telangiectasia-mutated and Rad3-related inhibitor elimusertib (BAY-1895344) in human plasma using LC-MS/MS.

Ataxia-telangiectasia-mutated and Rad3-related (ATR) is master regulator of the DNA-damage response that, through multiple mechanisms, can promote cancer cell survival in response to replication stress from sources, including chemotherapy and radiation. Elimusertib (BAY-1895344) is an orally available small-molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. To support these studies and define elimusertib pharmacokinetics, we developed a HPLC-MS method for its quantitation. A 50-μL volume of plasma was subjected to acetonitrile protein precipitation and then chromatographic separation using a Phenomenex Polar-RP column (2 × 50 mm, 4μm) and a gradient mobile phase consisting of 0.1% formic acid in acetonitrile and water during a 7-min run time. Mass spectrometric detection was achieved using a SCIEX 4000 triple-stage mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, the assay was linear from 30 to 5000 ng/mL and proved to be both accurate (93.5-108.2%) and precise (<6.3% coefficient of variation) fulfilling criteria from the Food and Drug Administration guidance on bioanalytical method validation. This LC-MS/MS assay will support several ongoing clinical studies by defining elimusertib pharmacokinetics.

DNA Repair Inhibitors Potentiate Fractionated Radiotherapy More Than Single-Dose Radiotherapy in Breast Cancer Cells.

Simple SummaryDNA damage response (DDR) inhibitors have been shown to sensitize cells to radiation yet have seen limited application in clinical settings. This could be due to a lack of understanding of how these inhibitors interact with ionizing radiation (IR) dose fractionation and cellular repair. Our study investigated the radiosensitizing effect of different DDR inhibitors on human breast cancer cells, utilizing single-dose and fractionated IR. Their effect on damage repair, DNA double-strand break repair kinetics and cell cycle distribution was also evaluated. The main finding was that radiosensitization by DDR inhibition was more prominent when combined with fractionated IR than single-dose IR. Moreover, DDR inhibition impeded the repair of IR-induced DNA double-strand breaks. Altogether, our study established the radiosensitizing potential of DDR inhibitors while highlighting the importance of IR dose fractionation in similar studies.Pharmacological inhibitors of DNA damage response (DDR) proteins, such as the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) kinases and poly (ADP-ribose) polymerase (PARP), have been developed to overcome tumor radioresistance. Despite demonstrating radiosensitization preclinically, they have performed suboptimally in clinical trials, possibly due to an incomplete understanding of the influence of DDR inhibition on ionizing radiation (IR) dose fractionation and sublethal damage repair. Hence, this study aimed to evaluate the radiosensitizing ability under fractionation of ATM inhibitor AZD0156, ATR inhibitor AZD6738 and PARP inhibitor AZD2281 (olaparib), utilizing MDA-MB-231 and MCF-7 human breast cancer cells. Clonogenic assays were performed to assess cell survival and sublethal damage repair after treatment with DDR inhibitors and either single-dose or fractionated IR. Immunofluorescence microscopy was utilized to evaluate DNA double-strand break repair kinetics. Cell cycle distributions were investigated using flow cytometry. All inhibitors showed significant radiosensitization, which was significantly greater following fractionated IR than single-dose IR. They also led to more unrepaired DNA double-strand breaks at 24 h post-IR. This study provides preclinical evidence for the role of AZD0156, AZD6738 and olaparib as radiosensitizing agents. Still, it highlights the need to evaluate these drugs in fractionated settings mirroring clinical practice to optimize the trial design.

Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance.

PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.