ATR Inhibitor AZD6738 Research Articles

Abstract A080: Olaparib and the ATR inhibitor AZD6738 in relapsed, refractory cancer patients with homologous recombination (HR) repair mutations – OLAPCO

Abstract Introduction: Olaparib is a PARP inhibitor (PARPi) that provides significant clinical benefit in several BRCA-mutant cancers, including ovarian, breast, pancreas and prostate. The benefit is reduced considerably in patients with multiple prior lines of therapy and olaparib resistance has no specific treatment. PARP inhibition results in replication stress (RS) due to unrepaired single strand DNA breaks (SSB) and PARP trapping in BRCA- and other HR repair-deficient tumors. ATR has critical roles in the cellular response to SSB and RS. This makes ATR inhibitors an attractive partner with olaparib, since ATR inhibition has the potential to reverse the two major mechanisms of PARP inhibitor resistance, including restored HR or stabilization of stalled replication forks. The OLAPCO trial (NCT02576444 clinicaltrials.gov) investigated. the combination of olaparib and AZD6738, an inhibitor of ATR, in relapsed, refractory cancer patients with tumors harboring HR repair mutations and in patients with BRCA-mutated PARPi pre-treated/resistant high-grade serous ovarian cancer (HGSOC). Methods: Patients with treatment-refractory, relapsed cancer were enrolled at 4 participating centers. Germline and somatic mutations had to be deleterious by COSMIC or ClinVar for eligibility. Performance status and organ function requirements were standard for early phase trials. Olaparib was given at 300 mg bid daily and AZD6738 at 160 mg daily days 1-7 in a 28-day cycle. Patients were treated until progression. Objective response was assessed by RECIST1.1 and toxicity was assessed by CTCAE4.0. Endpoints were confirmed complete [CR] or partial [PR] response rate and clinical benefit (CB) rate (response [PR] and stable disease [SD] for > 16 weeks). Results: We enrolled 24 patients; 17 (71%) females; median age 59 (36-78) years. Patients were heavily pretreated, with a median of 4 (0-10) prior regimens. Myelosuppression, especially anemia and thrombocytopenia, was the most frequent toxicity but no patient required discontinuation. Two patients required olaparib reductions for anemia. At the time of data cut-off, 20 patients are evaluable for response assessment. One of 5 patients with ATM mutations had a CR, 2 patients have CB ongoing at 12+ months. Of 7 patients with HGSOC resistant to platinum and PARP inhibitors (1-3 prior agents), 1 achieved a PR ( -90%), 3 had a best response of SD with regression < 30% (1 ongoing at 1 year) and 3 patients had progression (PD) as best response (Table 1). No other mutation or cancer type demonstrated objective response. Conclusions: The combination of olaparib and the ATR inhibitor AZD6738 demonstrated preliminary activity in patients with tumors harboring ATM mutations and in PARPi-resistant BRCA1/2-mutated HGSOC. Activity in ATM loss with an ATR inhibitor is consistent with the expected synthetic lethality of these interwoven DNA repair pathways. The encouraging data in HGSOC patients who have already progressed on a PARP inhibitor warrants additional study to further define the potential of this regimen to reverse PARPi resistance in BRCA-mutated HGSOC and other relevant solid tumors. The durability of responses in both groups (4 >1 year) is promising. Table 1MutationATMBRCA prostate pancreasBRCA Prior PARPi HGSOCCHEK2MUS81PALB2IDH and SDHD #5471142 CR1 PR0 1 SD > 4 mos213 11 PD 33112 Inevaluable110 11 Citation Format: Joseph Paul Eder, Davendra Sohal, Haider Mahdi, Khanh Do, Vicki Keedy, Navid Hafez, Deborah Doroshow, Manuel Avedissian, Peter Mortimer, Colin Glover, Patricia LoRusso, Juliane M Juergensmeier, Geoffrey I Shapiro. Olaparib and the ATR inhibitor AZD6738 in relapsed, refractory cancer patients with homologous recombination (HR) repair mutations – OLAPCO [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A080. doi:10.1158/1535-7163.TARG-19-A080

DNA Damage Response Inhibitor Combinations Exert Synergistic Antitumor Activity in Aggressive B-Cell Lymphomas

The DNA damage response (DDR) kinases ATR, Chk1, and Wee1 play vital roles in the response to replication stress and in maintaining cancer genomic stability. Inhibitors of these kinases are currently under clinical investigation. Mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) are aggressive lymphomas whose clinical outcome is still largely unsatisfactory. These cell lymphoma subtypes are highly dependent on both Chk1 and Wee1 for survival. We investigated the activity of the ATR inhibitor AZD6738 as single agent and in combination with either Chk1 (AZD6738) or Wee1 (AZD1775) inhibitors in several preclinical models of MCL and DLBCL. This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, and validation experiments on in vivo models. Cellular and molecular mechanisms of the observed synergistic effect as well as pharmacodynamic analysis of in vivo samples were studied. AZD6738 exerted a strong synergistic cytotoxic effect in combination with both AZD7762 and AZD1775 in the 2 lymphoma subtypes regardless of their TP53, MYC, and ATM mutational status. These DDR inhibitor combinations, similarly to the Chk1/Wee1 inhibitor combination, caused a marked S-phase delay, with an increase in cyclin-dependent kinases (CDK) activity, increased DNA damage, and decreases in Wee1, MYC, and RRM2 protein levels. The synergistic in vitro activity translated to striking in vivo antitumor activity. DDR-DDR inhibitor combinations could potentially offer promising novel therapeutic strategies for patients with B-cell lymphoma.

Development and Scale-up of a Route to ATR Inhibitor AZD6738

AZD6738 is currently being tested in multiple phase I/II trials for the treatment of cancer. Its structure, comprising a pyrimidine core decorated with a chiral morpholine, a cyclopropyl sulfoximin...

ATR Inhibition Potentiates the Radiation-induced Inflammatory Tumor Microenvironment.

ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment.Experimental Design: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy. Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT. We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid-sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Combined ATR and DNA-PK Inhibition Radiosensitizes Tumor Cells Independently of Their p53 Status.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of cancer deaths. Cisplatin-based chemoradiotherapy is a standard of care for locally advanced disease. ATR and DNA-PK inhibition (DNA-PKi) are actively being investigated in clinical trials with preclinical data supporting clinical translation as radiosensitizers. Here, we hypothesized that targeting both ATR and DNA-PK with small molecule inhibitors would increase radiosensitization of HNSCC cell lines. Radiosensitization was assessed by Bliss independence analysis of colony survival data. Strong cell cycle perturbing effects were observed with ATR inhibition reversing the G2/M arrest observed for radiation-DNA-PKi. Increased apoptosis in combination groups was measured by Sub-G1 DNA populations. DNA-PKi increased radiation-induced RAD51 and gamma-H2Ax foci, with the addition of ATR inhibition reducing levels of both. A sharp increase in nuclear fragmentation after aberrant mitotic transit appears to be the main driver of decreased survival due to irradiation and dual ATR/DNA-PKi. Dual inhibition of DNA-PK and ATR represents a novel approach in combination with radiation, with efficacy appearing to be independent of p53 status. Due to toxicity concerns, careful assessment is necessary in any future translation of single or dual radiosensitization approaches. Ongoing clinical trials into the ATR inhibitor AZD6738 plus radiation, and the phenotypically similar combination of AZD6738 and the PARP inhibitor olaparib, are likely to be key in ascertaining the toxicity profile of such combinations.

PO-261 Multimodal mass spectrometry imaging to understand drug delivery, metabolism, response and amp; resistance in pancreatic ductal adenocarcinoma

IntroductionThe desmoplastic Tumour MicroEnvironment (TME) in Pancreatic Ductal Adenocarcinoma (PDA) plays a key role in drug delivery, metabolism and resistance; drug interventions in turn regulate tumour metabolism. Mass Spectrometry Imaging (MSI) techniques may provide information about regional tumour metabolic profile and drug delivery to enhance our understanding of TME heterogeneity and its impact on drug efficacy and resistance.Material and methodsMultimodal MSI acquisition of the tissue distribution of gemcitabine, the ATR inhibitor AZD6738, their metabolites and the endogenous metabolome was performed in a KPC GEMM mouse model of PDA.Targeted analysis of the compounds and their metabolites were used to evaluate drug delivery. Small molecule quantitation including haem and metabolic markers such as ATP/ADP/AMP, were used together with H and E and staining for known molecular biomarkers including Pan-CK and αSMA to characterise tissue architecture.Untargeted analyses including statistical identification of discriminative or colocalised metabolites were used to identify de novo endogenous metabolite biomarkers and cellular phenotypes driving tumour heterogeneity.Results and discussionsMSI revealed significant intratumoural heterogeneity of drug delivery and drug metabolism. Highest delivery of the parent compounds (AZD6738 and dFdC) were found to colocalise with haem in areas confirmed histologically to be necrotic and haemorrhagic. dFdC metabolism appeared related to TME metabolic heterogeneity. The active and inactive metabolites of gemcitabine (including dFdCTP and dFdU) demonstrated differential distribution, both from the parent compounds and each other in the tissue.Unsupervised clustering based segmentation and colocalisation analysis of the MSI metabolomic data enabled identification and characterisation of these distinct tissue regions based on similarities in their metabolic profile.ConclusionWe have shown that MSI allows spatial resolution of drug delivery, metabolism and that MSI-based metabolomics analysis enables detection of greater tumour heterogeneity than visible by traditional pathology methods such as H and E. Combining this with biomarker information from Imaging Mass Cytometry (IMC) may enable identification of the cell types and phenotypes responsible for the differential metabolic effects observed with combination therapies. Innovative, information-rich technologies such as IMC and MSI may drive greater understanding of the impact of tumour heterogeneity on drug efficacy in vivo and ultimately in patients.

Abstract 2852: Identifying circulating biomarkers of acute response and resistance to clinical ATR and Chk1 inhibitors

Abstract Objectives: Clinical ATR kinase inhibitors (ATRi) are an emerging class of therapeutics being used to treat diverse solid tumor malignancies, including ovarian cancers. The development of circulating biomarkers indicating response or resistance to clinical ATRi will support the ongoing development of this emerging class of therapeutics. This study is focused on establishing isogenic models of ATRi-resistance in ovarian cancer cell lines and identifying conserved secreted protein alterations correlating with acute response or resistance to ATRi treatment in these models. Methods: We generated isogenic cell line models of ATRi-resistant, CCNE1-amplified (OVCAR3) and non-CCNE1 amplified (OV90) ovarian cancer cell by metronomic treatment of cell lines with the ATR inhibitor AZD6738 (Astra Zeneca). We characterized sensitivity of models to AZD6738 as well as inhibitors of checkpoint kinase 1 (Chk1, LY2606368), ataxia-telangiectasia mutated (ATM, KU55933) poly (ADP-ribose) polymerase (PARP, BMN-673) and cisplatin combination by dose response assay and further assessed impact of ATRi treatment on cell cycle. Lastly, we analyzed conditioned media by LC-MS/MS-based proteomic analyses from ATRi-resistant OVCAR3 cells or following acute treatment with ATRi. Results: Metronomic treatment of ovarian cancer cells with ATRi induced resistance to ATRi irrespective of CCNE1 status. ATRi-resistant cells were also resistant to Chk1i, but not to ATM, PARP inhibitors or combinations of ATRi and cisplatin. Cell cycle analyses revealed that ATRi-sensitive cells arrest in S-phase and continue active DNA replication, whereas ATRi-resistant cells arrest in G1/S phase and cease DNA replication following ATRi treatment. Comparison of proteins identified in conditioned media revealed > 30 proteins bearing signal peptide sequences potentially mediating cellular secretion as significantly elevated in the cellular secretomes of ATRi-resistant or ATRi sensitive cells treated with ATRi, with only a single protein co-altered between these sample, i.e. alpha-N-acetylgalactosaminidase (NAGA). Conclusions: Our findings show that metronomic treatment of ovarian cancer cells with ATR inhibitors induces resistance to ATRi as well as an inhibitor of Chk1, an immediate downstream effector of ATR. Our analyses show that ATRi-resistant cells remain sensitive to inhibitors of orthogonal DNA damage response signaling pathways, i.e. ATM and PARP, as well as to cisplatin sensitization induced by ATRi co-treatment. ATRi-resistant cells further exhibit a conserved G1/S-phase cell cycle arrest response to ATRi treatment. Lastly, our preliminary secretome analyses provide proof of concept data that identification of secreted biomarkers specific for acute response or resistance to ATRi (Chk1i)-treatment is feasible. Citation Format: Nicholas Bateman, Wei Ao, Domenic Tommarello, Kelly Conrads, Pang-ning Teng, Kathleen Darcy, Chad Hamilton, G. Larry Maxwell, Christopher Bakkenist, Thomas Conrads. Identifying circulating biomarkers of acute response and resistance to clinical ATR and Chk1 inhibitors [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 2852.

BRD4 facilitates replication stress-induced DNA damage response

Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. Here we describe a novel role for BRD4 in DNA damage response (DDR). BRD4 associates with and regulates the function of pre-replication factor CDC6 and plays an indispensable part in DNA replication checkpoint signaling. Inhibition of BRD4 by JQ1 or AZD5153 resulted in a rapid, time-dependent reduction in CHK1 phosphorylation and aberrant DNA replication re-initiation. Furthermore, BRD4 inhibition sensitized cancer cells to various replication stress-inducing agents, and synergized with ATR inhibitor AZD6738 to induce cell killing across a number of cancer cell lines. The synergistic interaction between AZD5153 and AZD6738 is translatable to in vivo ovarian cell-line and patient-derived xenograft models. Taken together, our study uncovers a new biological function of BRD4 and provides mechanistic rationale for combining BET inhibitors with DDR-targeted agents for cancer therapy.

CYP-Mediated Sulfoximine Deimination of AZD6738.

In hepatic S9 and human liver microsomes (HLMs) the sulfoximine moiety of the ATR inhibitor AZD6738 is metabolized to its corresponding sulfoxide (AZ8982) and sulfone (AZ0002). The initial deimination to AZ8982 is nominally a reductive reaction, but in HLMs it required both NADPH and oxygen and also was inhibited by 1-aminobenzotriazole at a concentration of 1 mM. Studies conducted in a panel of 11 members of the cytochrome P450 (P450) family (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 CYP2J2, CYP3A4, and CYP3A5) confirmed that deimination was an oxidative process that was mediated largely by CYP2C8 with some CYP2J2 involvement, whereas the subsequent oxidation to sulfone was carried out largely by CYP2J2, CYP3A4, and CYP3A5. There was no measureable metabolism in flavin-containing monooxygenase (FMO) enzymes FMO3, FMO5 or NADPH cytochrome C reductase. Studies using Silensomes, a commercially available HLM in which specific members of the P450 family have been inhibited by selective mechanism-based inhibitors, showed that when CYP2C8 was inhibited, the rate of deimination was reduced by 95%, suggesting that CYP2J2 is only playing a minor role in HLMs. When CYP3A4 was inhibited, the rate increased by 58% due to the inhibition of the subsequent sulfone formation. Correlation studies conducted in HLM samples from different individuals confirmed the role of CYP2C8 in the deimination over CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. Hence, although nominally a reduction, the deimination of AZD6738 to its sulfoxide metabolite AZ8982 is an oxidation mediated by CYP2C8, and this metabolite is subsequently oxidized to the sulfone (AZ0002) largely by CYP3A.

Defining DDR defectiveness in pancreatic cancer.

4115 Background: Recent whole genome sequencing analysis of Pancreatic Cancer (PC) revealed that up to 24% of PC may harbor defects in DNA damage response (DDR). There is increasing evidence that DDR defective tumors preferentially respond to DNA damaging agents, representing novel therapeutic strategy for PC using a synthetic lethality approach. The aim of this study is to define and refine DDR defective phenotypes in PC using next-generation preclinical model systems. Methods: From a panel of 40 patient-derived cell lines (PDCL) and 64 patient-derived xenografts (PDX), generated and extensively characterized as part of the International Cancer Genome Initiative (ICGC), we identified DDR defective models using recently described putative biomarkers of DDR defectiveness. Cytotoxic viability assays were performed using a panel of DNA damaging agents and inhibitors of key molecules in DDR pathway, including Cisplatin, PARP inhibitors, ATR inhibitor (AZD6738); and ATM inhibitor (AZD0156). Appropriate subcutaneous PDX models were also generated to test the hypothesis using various therapeutic regimens. Results: DDR defective PDCLs were selected based on a combination of an unstable genome, and/or a high BRCA mutational signature, and/or deleterious mutations in BRCA1/2, PALB2 or ARID1A. DDR defective PDCLs were significantly more sensitive to Cisplatin, PARP and ATR inhibitors. The ATR inhibitor AZD6738, and ATM inhibitor AZD0156 sensitized PDCLs with no putative biomarkers of DDR defectiveness to Cisplatin, demonstrating a ‘fabricated’ synthetic lethality. A BRCA1 mutant PDX model responded exceptionally to Cisplatin and the PARP inhibitor Olaparib monotherapy. Conclusions: This study provides proof of concept data that DDR deficiency represents an attractive segment to target in PC using a variety of DNA damaging agents and novel agents targeting key molecules of the DDR pathway in PC. In addition, the DDR defective segment may be significantly larger than just germline BRCA1/2 mutants, which is current clinical trial recruitment criteria. Robust molecular assays with clinical utility to define DDR defectiveness is urgently needed.

LC–MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR inhibitor AZD6738 in mouse plasma

The ATM kinase inhibitor AZ31 and ATR kinase inhibitor AZD6738 are in various phases of preclinical and clinical evaluation for their ability to potentiate chemoradiation. To support the preclinical evaluation of their pharmacokinetics, we developed and validated an LC–MS/MS assay for the simultaneous quantification of AZ31 and AZD6738 in mouse plasma. A “dilute and shoot” method was used to precipitate proteins from a sample volume of 50μL. Chromatographic separation was achieved using a Phenomenex Polar-RP column and a gradient mobile phase consisting of methanol–water with 0.1% formic acid. Detection was accomplished using a Waters Quattro Micro mass spectrometer in positive ionization mode. The assay utilizing 50μL sample was linear from 10 to 5000ng/mL and determined to be both accurate (−8.2 to 8.6%) and precise (<5.4% CV) and achieved the criteria for U.S. FDA guidance for bioanalytical method validation. Quantification was achieved in mouse tissue homogenate using a separate 200μL sample preparation. This LC–MS/MS assay will be essential for determining the tissue distribution and pharmacokinetics in future mouse studies.

Radiosensitization by the ATR Inhibitor AZD6738 through Generation of Acentric Micronuclei.

AZD6738 is an orally active ATR inhibitor (ATRi) currently in phase I clinical trials. We found in vitro growth inhibitory activity of this ATRi in a panel of human cancer cell lines. We demonstrated radiosensitization by AZD6738 to single radiation fractions in multiple cancer cell lines independent of both p53 and BRCA2 status by the clonogenic assay. Radiosensitization by AZD6738 to clinically relevant doses of fractionated radiation was demonstrated in vitro using a 3D tumor spheroid model and, in vivo, AZD6738 radiosensitized by abrogating the radiation-induced G2 cell-cycle checkpoint and inhibiting homologous recombination. Mitosis with damaged DNA resulted in mitotic catastrophe as measured by micronucleus formation by live-cell fluorescent-ubiquitination cell-cycle imaging of cell-cycle progression and nuclear morphology. Induction of micronuclei was significantly more prominent for AZD6738 compared with inhibition of the downstream kinase CHK1 alone at isoeffective doses. Micronuclei were characterized as acentric chromosomal fragments, which displayed characteristics of increased DNA damage and cell-cycle dyssynchrony when compared with the primary nucleus. Mol Cancer Ther; 16(1); 25-34. ©2016 AACR.

Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738.

Understanding the therapeutic effect of drug dose and scheduling is critical to inform the design and implementation of clinical trials. The increasing complexity of both mono, and particularly combination therapies presents a substantial challenge in the clinical stages of drug development for oncology. Using a systems pharmacology approach, we have extended an existing PK-PD model of tumor growth with a mechanistic model of the cell cycle, enabling simulation of mono and combination treatment with the ATR inhibitor AZD6738 and ionizing radiation. Using AZD6738, we have developed multi-parametric cell based assays measuring DNA damage and cell cycle transition, providing quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model is predictive of tumor growth observed in in vivo mouse xenograft studies. The model is being used for phase I clinical trial designs for AZD6738, with the aim of improving patient care through quantitative dose and scheduling prediction.

Abstract LB-107: Targeting ATR using a novel ATR inhibitor AZD6738 in human gastric cancer cells

Abstract Introduction: The DNA repair system is critical for maintaining genomic integrity. In particular, the homologous recombination (HR) repair pathway, on that repairs DNA double-strand breaks (DSBs), is directly associated with cancer development. Many cancer cells could contain mutations in genes involved in the HR pathway including BRCA1/2, ATM, ATR, and RAD51. ATR can be activated by various types of DNA damage and it initiates DNA damage-induces signaling cascade. As a result, ATR pathway components are considered promising therapeutic targets because ATR inhibition is likely to have greater deleterious effect on cancer cells. Materials and Methods: The antiproliferative activity of AZD6738 was examined in vitro using a cytotoxic assay, cell cycle analysis, and western blotting. To figure out the action mechanism of AZD6738 in gastric cancer cells, the expression of proteins which participate in DNA repair were investigated. The accumulation of DNA damage was also assessed with a DNA comet assay. These in vitro data were validated in vivo using a human gastric cancer xenograft model. Results: Human gastric cancer cell lines showed heterogeneous response to AZD6738. In AZD6738 sensitive cells, ATR inhibition leads to the accumulation of unrepaired DSBs due to dysfunctional RAD51 foci formation along with increased caspase 3-dependent cell death and S phase cell cycle arrest. Compared with sensitive cells, the activation of ATM-chk2 signaling pathway under the ATR inhibition was observed in the AZD6738 insensitive cells. It suggests that the activation of ATM-chk2 signal leads to attenuation of AZD6738 sensitivity. Furthermore, AZD6738 significantly suppressed tumor growth with increased apoptosis in vivo. Conclusion: We evaluated the anti-tumor activity of AZD6738 in gastric cancer in vitro and in vivo model. This is the first study to show that AZD6738 interferes with RAD51-mediated homologous recombination, as well as promotes cell death by inducing S cell cycle arrest and apoptosis in gastric cancer cells. Our findings can help to promote novel treatment strategies using AZD6738 in gastric cancer. Citation Format: Ahrum Min, Seock-Ah Im, Hyemin Jang, Seongyeong Kim, Miso Lee, Jungeun Kim, Kyung-Hun Lee, Sae-Won Han, Tae-Young Kim, Do-Youn Oh, Tae-You Kim, Woo-Ho Kim, Yung-Jue Bang. Targeting ATR using a novel ATR inhibitor AZD6738 in human gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-107. doi:10.1158/1538-7445.AM2015-LB-107

ATR Inhibition Exacerbates Replication Stress in TP53 or ATM Deficient CLL Cells and Enhances Sensitivity to Chemotherapy and Targeted Therapy

DNA damage response (DDR) defects, particularly TP53 or biallelic ATM aberrations, are associated with chemoresistance in chronic lymphocytic leukemia (CLL). Chemoimmunotherapy or B-cell receptor signaling inhibitors alone may not be sufficient to overcome adverse prognosis or provide durable response in TP53 or biallelic ATM inactivated CLL. In particular, genomic instability resulting from impaired DDR facilitates rapid clonal evolution leading to treatment refractoriness or disease relapse. Development of therapeutic approaches specifically targeting DDR defects is therefore necessary for effective long-term control of DDR-defective CLL.We previously demonstrated selective cytotoxicity of the ATR inhibitor AZD6738 towards TP53 or ATM null CLL cells, and validated this in CLL xenograft models for biallelic TP53 or ATM loss. Here, we provide mechanistic insight into the synthetically lethal interactions between ATR pathway inhibition and TP53 or ATM loss in CLL, and offer experimental evidence supporting the use of ATR inhibition in combination with conventional chemotherapies and other targeted therapies in CLL.To determine the mechanism of ATR inhibition in CLL, we first investigated its effect on DDR-intact cycling primary cells. We observed compensatory activation of the ATM/p53 pathway in AZD6738-treated cells in response to hydroxyurea, providing evidence for crosstalk between ATR and ATM/p53 pathways. Next, we explored the cellular consequence of ATR inhibition in CLL cells with DDR loss. To determine the impact of ATR inhibition on DNA replication, we performed DNA fiber analysis, which revealed significantly increased replication fork stalling and firing of replication origins upon AZD6738 treatment in ATM/p53-defective CLL. To investigate the effect of ATR inhibition on DNA damage, we measured γH2AX and 53BP1 foci formation, markers of DNA double-strand breaks. Significant induction of γH2AX and 53BP1 foci was seen in ATM/p53-defective CLL cells upon AZD6738 treatment, suggesting that ATR inhibition exacerbates replication stress in ATM/p53 defective cells by imposing requirement for DDR through the ATM/p53 pathway.On the contrary, 53BP1 bodies, a marker of unreplicated DNA, appeared in ATM/p53 proficient but not in ATM/p53 deficient cells following AZD6738 exposure, indicating that cell cycle arrest in response to replication stress is ATM/p53 dependent. This was corroborated by cell cycle profiling and co-labeling experiments of γH2AX with phosphohistone H3 ser10, a marker of mitosis, showing that ATM/p53-defective CLL cells carrying unrepaired DNA damage continued to cycle. Thus, ATR inhibition results in accumulation of intolerable levels of DNA damage in DDR-defective CLL, leading to cell death by mitotic catastrophe, which we have confirmed both in vitro and in AZD6738-treated murine xenograft models.The benefit of ATR inhibition as a therapeutic strategy for DDR-defective CLL lies in its ability to alter the subclonal landscape in favor of less unstable DDR-proficient subclones, which are less susceptible to clonal evolution, thus reducing the likelihood of disease relapse. We reasoned that this provides a strong rationale for addition of AZD6738 to existing therapeutic agents for the treatment of CLL with DDR defects. We demonstrated synergistic and additive effects of low-dose AZD6738 in ATM/p53-defective CLL cells with DNA damaging agents such as chlorambucil, fludarabine, bendamustine and cyclophosphamide, BCR-signaling inhibitors and the PARP inhibitor olaparib. We validated the AZD6738 plus chlorambucil combination in biallelic TP53 or ATM inactivated primary CLL xenografts, where combined AZD6738/chlorambucil treatment was superior to chlorambucil alone, as evidenced by significantly greater reduction in tumor load and percentage of CLL subclones with del(17p) or del(11q) in animals treated with combination regimen compared to single-agent chlorambucil. Similar investigations evaluating AZD6738 plus ibrutinib versus ibrutinib monotherapy in primary CLL xenografts are currently underway.We conclude that ATR inhibition is a suitable approach for targeting the loss of p53 function in aggressive CLL subclones and should be considered as a valuable addition to DNA damaging agents and current targeted treatments. DisclosuresOff Label Use: ATR inhibitor AZD6738 targets TP53-null or ATM-null phenotype inducing synthetic lethality. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

Abstract 5485: Induction of proliferation sensitizes chronic lymphocytic leukemia cells to apoptosis mediated by the ATR inhibitor AZD6738

Abstract ATR (Ataxia Telangiectasia and RAD3-related) is a PI3K-related kinase involved in repair of DNA damage incurred through the genotoxic stress of cellular replication. During cell division, ATR is recruited and activated at sites of single strand DNA breaks, such as those that occur at stalled replication forks, and helps initiate DNA repair. Tumor cells may develop an enhanced dependency on ATR to maintain genomic stability and survival, making ATR an attractive target for anticancer therapy. AZD6738 is a first-in-class small molecule that inhibits ATR at sub micromolar (uM) concentrations. We examined the leukemic cells from patients with chronic lymphocytic leukemia (CLL) for expression of ATR and sensitivity to this specific ATR inhibitor. For this, we examined both resting CLL cells and CLL cells induced to undergo replication, as assessed by flow cytometry of CLL cells stained with Carboxyfluorescein succinimidyl ester (CFSE). We found that conditions that induced of leukemic cell proliferation (eg: CD40 ligation in the context of interleukin(IL)4 and IL10) also induced expression of ATR. Examination of both resting and proliferating leukemic cells for sensitivity to AZD6738 revealed that those leukemic cells that expressed ATR were highly sensitive to the cytotoxic effects of this ATR inhibitor in a time and dose dependent fashion. As such, concentrations of AZD6738 as low as 1 uM were cytotoxic for proliferating CLL cells. In contrast, the IC50 of resting CLL cells was approximately 30 uM, indicating that resting CLL cells are relatively insensitive to the activity of this drug. AZD6738 may therefore be particularly effective in eliminating the leukemic cells that are being stimulated in the leukemia microenvironment found in lymphoid tissues, which provides survival/growth signals that ordinarily mitigate the activity of standard cytotoxic drugs. ATR inhibition also may be particularly effective in tumors lacking ATM (Ataxia Telangiectasia Mutated). Such cells are even more dependent on the activity of ATR to repair DNA damage that occurs during replication or genotoxic stress, such as that incurred by cancer chemotherapy. To this end, we are examining differential sensitivity to ATR inhibition of leukemic cells that lack functional ATM, which is often times associated with deletions of the long arm of chromosome 11 (Del 11q22). An ongoing phase 1 clinical trial testing the safety of AZD6738 in patients with relapsed or refractory CLL with Del11q also will examine the activity of this compound for the treatment of patients with CLL. Citation Format: Michael Y. Choi, Jessie-Farah Fecteau, Jeff Brown, Alan Lau, Thomas J. Kipps. Induction of proliferation sensitizes chronic lymphocytic leukemia cells to apoptosis mediated by the ATR inhibitor AZD6738. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2014-5485

Synthetic Lethality In CLL With DNA Damage Response Defect By Targeting ATR Pathway

▪Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed.ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality' and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM.First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea.We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support.To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss.We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures:Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.