ATR Inhibitor AZD6738 Research Articles

Discovery of a Meisoindigo-Derived PROTAC as the ATM Degrader: Revolutionizing Colorectal Cancer Therapy via Synthetic Lethality with ATR Inhibitors.

Meisoindigo (Mei) has long been recognized in chronic myeloid leukemia (CML) treatment. To elucidate its molecular target and mechanisms, we embarked on designing and synthesizing a series of Mei-derived PROTACs. Through this endeavor, VHL-type PROTAC 9b was identified to be highly cytotoxic against SW620, SW480, and K562 cells. Employing DiaPASEF-based quantitative proteomic analysis, in combination with extensive validation assays, we unveiled that 9b potently and selectively degraded ATM across SW620 and SW480 cells in a ubiquitin-proteasome-dependent manner. 9b-induced selective ATM degradation prompted DNA damage response cascades, thereby leading to the cell cycle arrest and cell apoptosis. This pioneering discovery renders the advent of ATM degradation for anti-cancer therapy. Notably, 9b-induced ATM degradation synergistically enhanced the efficacy of ATR inhibitor AZD6738 both in vitro and in vivo. This work establishes the synthetic lethality-inducing properties of ATR inhibitors in the ATM-deficient context, thereby providing new avenues to innovative therapies for colorectal cancer.

Metronomic dosing of ovarian cancer cells with the ATR inhibitor AZD6738 leads to loss of CDC25A expression and resistance to ATRi treatment

ObjectiveATR kinase inhibitors promote cell killing by inducing replication stress and through potentiation of genotoxic agents in gynecologic cancer cells. To explore mechanisms of acquired resistance to ATRi in ovarian cancer, we characterized ATRi-resistant ovarian cancer cells generated by metronomic dosing with the clinical ATR inhibitor AZD6738. MethodsATRi-resistant ovarian cancer cells (OVCAR3 and OV90) were generated by dosing with AZD6738 and assessed for sensitivity to Chk1i (LY2603618), PARPi (Olaparib) and combination with cisplatin or a CDK4/6 inhibitor (Palbociclib). Models were characterized by diverse methods including silencing CDC25A in OV90 cells and assessing impact on ATRi response. Serum proteomic analysis of ATRi-resistant OV90 xenografts was performed to identify circulating biomarker candidates of ATRi-resistance. ResultsAZD6738-resistant cell lines are refractory to LY2603618, but not to Olaparib or combinations with cisplatin. Cell cycle analyses showed ATRi-resistant cells exhibit G1/S arrest following AZD6738 treatment. Accordingly, combination with Palbociclib confers resistance to AZD6738. AZD6738-resistant cells exhibit altered abundances of G1/S phase regulatory proteins, including loss of CDC25A in AZD6738-resistant OV90 cells. Silencing of CDC25A in OV90 cells confers resistance to AZD6738. Serum proteomics from AZD6738-resistant OV90 xenografts identified Vitamin D-Binding Protein (GC), Apolipoprotein E (APOE) and A1 (APOA1) as significantly elevated in AZD6738-resistant backgrounds. ConclusionsWe show that metronomic dosing of ovarian cancer cells with AZD6738 results in resistance to ATR/ Chk1 inhibitors, that loss of CDC25A expression represents a mechanism of resistance to ATRi treatment in ovarian cancer cells and identify several circulating biomarker candidates of CDC25A low, AZD6738-resistant ovarian cancer cells.

Abstract P2-17-03: Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models

Abstract Background: Triple negative breast cancer (TNBC), defined by negative hormone receptor (HR) status and the lack of HER2 gene amplification, is a significant clinical challenge because of its aggressive clinical course and association with a poor prognosis. Recent studies demonstrated that approximately a third of TNBC has low expression of HER2, so called HER2-low TNBC, which could be targeted by the new generation HER2-directed antibody-drug conjugate (ADC). Trastuzumab deruxtecan (also known as T-Dxd, DS-8201a) is a novel HER2-directed ADC with a topoisomerase 1 inhibitor payload and a tetrapeptide-based cleavable linker, which has been FDA approved for patients with metastatic HER2-positive, or amplified breast cancer. T-Dxd has also shown to improve survival outcomes when compared to physicians’ choice chemotherapy in patients with metastatic HR-negative/HER2-low breast cancer in the Phase 3 DESTINY-Breast04 trial. However, resistance is invariable. We hypothesize that small molecule inhibitors against DNA damage response (DDR) pathways could potentiate the DNA damaging effect of T-Dxd and improve its anti-tumor activity in HER2-low TNBC. Method: We conducted preclinical experiments to assess the anti-tumor activity of T-Dxd and each of the DDR pathway inhibitors, including the ATR inhibitor AZD6738, the ATM inhibitor AZD1390, and the PARP inhibitor olaparib, as well as the Wee 1 inhibitor AZD1775, either alone or in combination, compared to the vehicle treatment, in a panel of HER2-low TNBC PDX models in vivo, as well as mechanistic studies using HER2-low TNBC cell lines in vitro. HER2-low was defined as 1+ or 2+ on HER2 immunohistochemistry. Results: Four HER2-low TNBC PDX models, including WHIM2 (TP53 mutated, basal-like), WHIM6 (TP53 WT, basal-like), WHIM12 (TP53, PIK3CA, and PTEN mutated, claudin-low), and WHIM30 (BRCA1 and TP53 mutated, basal-like), as well as a panel of 4 TNBC cell lines with variable HER2 expression were included in the study. We demonstrated that the addition of DDR pathway inhibitors to T-Dxd led to further tumor shrinkage and delayed tumor progression in HER2-low TNBC PDX models in vivo and induced synergistic anti-tumor effects in vitro. Enhanced DNA damaging effect and apoptosis induction were observed with combination therapies in vitro. Biomarker analysis on xenograft tumors harvested 3 days following treatment with either Vehicle, T-Dxd, olaparib, or T-Dxd plus olaparib in WHIM6, was also performed, which demonstrated that the addition of olaparib enhanced DNA damage (increased pH2AX), and promoted apoptosis (increased cleaved PARP). Further mechanistic studies in HER2-low TNBC cell lines demonstrated the formation of DNA topoisomerase 1 covalent complexes (TopoIcc) following treatment with T-Dxd. Interestingly, while the formation of Topolcc was reduced with the addition of either ATR or ATM inhibitor, no reduction was observed with the addition of olaparib. Additionally, we observed that combination therapies reduced cell migration in vitro. The combination of olaparib with T-Dxd, in particular, reduced mammosphere formation and Epithelial Mesenchymal Transition (EMT) modulation markers, including SNAIL, SLUG and/or TWIST. Conclusion: Our study demonstrated that the combination of T-Dxd and DDR pathway inhibitors induced additive or synergistic anti-tumor effect in HER2-low TNBC. Our data from the studies of cell lines and PDX models provided preclinical rationale for the combination of T-Dxd and DDR pathway inhibitors in patients with HER2-low TNBC. The olaparib combination showed unique mechanistic characteristics compared with other DDR inhibitors, which are being further examined in additional HER2-low TNBC models. Citation Format: Adrian Gonzalez-Gonzalez, Zhanfang Guo, Alice Meroni, Emily Cybulla, Jeremy Hoog, Alessandro Vindigni, Cynthia Ma. Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-03.

The schedule of ATR inhibitor AZD6738 can potentiate or abolish antitumor immune responses to radiotherapy.

Inhibitors of the DNA damage signaling kinase ATR increase tumor cell killing by chemotherapies that target DNA replication forks but also kill rapidly proliferating immune cells including activated T cells. Nevertheless, ATR inhibitor (ATRi) and radiotherapy (RT) can be combined to generate CD8+ T cell-dependent antitumor responses in mouse models. To determine the optimal schedule of ATRi and RT, we determined the impact of short-course versus prolonged daily treatment with AZD6738 (ATRi) on responses to RT (days 1-2). Short-course ATRi (days 1-3) plus RT caused expansion of tumor antigen-specific, effector CD8+ T cells in the tumor-draining lymph node (DLN) at 1 week after RT. This was preceded by acute decreases in proliferating tumor-infiltrating and peripheral T cells and a rapid proliferative rebound after ATRi cessation, increased inflammatory signaling (IFN-β, chemokines, particularly CXCL10) in tumors, and an accumulation of inflammatory cells in the DLN. In contrast, prolonged ATRi (days 1-9) prevented the expansion of tumor antigen-specific, effector CD8+ T cells in the DLN, and entirely abolished the therapeutic benefit of short-course ATRi with RT and anti-PD-L1. Our data argue that ATRi cessation is essential to allow CD8+ T cell responses to both RT and immune checkpoint inhibitors.

ARID1A deficiency as a biomarker for sensitivity to radiation and ATR inhibition (232)

<b>Objectives:</b> <i>ARID1A,</i> which encodes a protein that participates in the SWI/SNF chromatin remodeling complex, is frequently mutated in ovarian and endometrial malignancies. The emergence of the role of <i>ARID1A</i> in gynecologic cancer progression reveals potential implications for therapeutic targets. Our primary purpose was to investigate <i>ARID1A</i> deficiency as a potential biomarker conferring sensitivity to ionizing radiation and ATR inhibitors. <b>Methods:</b> Paired isogenic cell lines ES2 and HCT116 were used containing wild-type <i>ARID1A</i> or homozygous loss of function <i>ARID1A</i> mutations. Ionizing radiation doses were delivered using Precision X-ray 320-kV orthovoltage unit at a dose rate of 2 Gy/45 seconds. Cytotoxicity assays were performed to evaluate cell survival following various doses of ATR inhibitor AZD6738. Clonogenic survival assays were performed with or without 50 nM of AZD6738 one hour prior to radiation at doses of 0, 1, 2, 4, or 6 Gy. Survival curves were generated and compared using the linear quadratic equation on GraphPad Prism. gH2AX foci formation following radiation at 5 Gy was observed using immunofluorescence (IF) and Western blot. IF images were analyzed using ImageJ. Cell cycle distribution following radiation at 5 Gy was performed using flow cytometry, and analysis was performed using FlowJo software. <b>Results:</b> <i>ARID1A</i>-deficient cells exhibited greater sensitivity to radiation alone compared to wild-type cells as measured by clonogenic survival and demonstrated a diminished DNA damage response following radiation compared to wild-type cells, as measured by gH2AX foci formation by IF and Western blot. <i>ARID1A</i>-deficient cells exhibited an impaired G2/M cell cycle checkpoint following radiation, as exhibited by diminished arrest at G2/M compared to wild-type cells. <i>ARID1A</i>-deficient cells demonstrated sensitization to AZD6738 alone compared to wild-type cells by cytotoxicity assay. Preliminary results suggest that <i>ARID1A-</i>deficient cells have greater sensitization to AZD6738 treatment prior to radiation compared to radiation alone. Further evaluation of combinatorial strategies is in progress. <b>Conclusions:</b> Our <i>in vitro</i> findings confirm that <i>ARID1A</i>-deficiency confers sensitivity to radiation and ATR inhibition, suggesting a biologically informed treatment strategy for <i>ARID1A</i>-deficient malignancies. Further work regarding dosing and timing of combination treatment will be explored in the future.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1β [IL-1β], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409’s efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Ataxia telangiectasia and Rad3-related inhibition by AZD6738 enhances gemcitabine-induced cytotoxic effects in bladder cancer cells.

The ataxia telangiectasia and rad3-related-checkpoint kinase 1 (ATR-CHK1) pathway is involved in DNA damage responses in many cancer cells. ATR inhibitors have been used in clinical trials in combination with radiation or chemotherapeutics; however, their effects against bladder cancer remain unclear. Here, the efficacy of combining gemcitabine with the novel ATR inhibitor AZD6738 was investigated in vitro in three bladder cancer cell lines (J82, T24, and UM-UC-3 cells). The effects of gemcitabine and AZD6738 on cell viability, clonogenicity, cell cycle, and apoptosis were examined. The combined use of gemcitabine and AZD6738 inhibited the viability and colony formation of bladder cancer cells compared to either treatment alone. Gemcitabine (5 nM) and AZD6738 (1 μM) inhibited cell cycle progression, causing cell accumulation in the S phase. Moreover, combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin V-positive cells, indicating apoptosis induction. Mechanistic investigations showed that AZD6738 treatment inhibited the repair of gemcitabine-induced double-strand breaks by interfering with CHK1. Combining AZD6738 with gemcitabine could therefore be useful for bladder cancer therapy.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Whole‐exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets

Whole‐exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets

EXTH-76. DEVELOPING THE NOVEL COMBINATION THERAPY OPTIONS FOR CANCER THERAPY USING TUMOR TREATING FIELDS TOGETHER WITH THE CHEMO AGENTS TARGETING THE REPLICATION STRESS PATHWAY

Abstract Tumor Treating Fields (TTFields) as a component of cancer therapy has been shown to provide significant clinical benefit. The disruption of mitosis was identified as the first mechanism of action, however, a novel role for TTFields outside of mitosis where TTFields downregulates the Fanconi’s Anemia genes and proteins results in replication stress and reduced DNA repair capacity. Given these results we hypothesized that TTFields would increase the sensitivity of tumor cells to chemotherapy agents that increase replication stress. An analysis of agents that target replication stress in different ways was initiated. PARP1: Targeting PARP1 protects DNA breaks by recruiting DNA repair and checkpoint proteins to the sites of damage. Using the PARP1 inhibitor olaparib followed by radiation resulted in synergistic cell killing compared to radiation or olaparib alone or in combination. Etoposide: Etoposide forms a ternary complex with topoisomerase II and prevents re-ligation of DNA strands to elicit DNA strand breaks and replication stress. When combined with TTFields cell killing increased synergistically vs. etoposide alone. AZD6738: ATR is an essential kinase regulator of the replication checkpoint that plays a critical role in safeguarding genome integrity from replication stress. The advantage of combining TTFields with the ATR inhibitor-AZD6738 was tested for cell killing and the combination vs. AZD6738 was found to be synergistic. Irinotecan: Irinotecan traps topoisomerase I- DNA in a ternary cleavage complex and inhibits the initial cleavage reaction and re-ligation steps resulting in increased replication stress. Irinotecan and TTFields was tested and found to be highly effective at tumor cell killing. Collectively, our results suggest that it is likely of considerable clinical benefit to combine TTFields with chemotherapy agents that cause replication stress. This notion may explain the results of a number of clinical trials and suggests that there may be novel TTFields/replication stress-inducing chemotherapy agent combinations worth exploring.

CSIG-19. DISRUPTION OF DNA DAMAGE RESPONSE MODULATES THE EFFICACY OF LOCAL IMMUNOTHERAPIES IN EXPERIMENTAL GLIOMA

Abstract RATIONALE Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR). METHODS We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF. RESULTS The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors. CONCLUSION DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.

Tumor Treating Fields Synergistically Enhances Cell Death in Non-Small Cell Lung and Pancreatic Cancer Cells Treated With the Combination of Radiation and Chemo Agents That Interfere With DNA Replication Fork Stability

<h3>Purpose/Objective(s)</h3> TTFields is a novel non-invasive physical modality of cancer therapy which was initially thought to interfere with cancer cell mitosis to induce cell death. Genomics and proteomic results suggested the additional mechanisms of action underlying TTFields induced cell death as increased DNA replication stress and DNA damage while decreasing DNA repair through the downregulation of Fanconi's Anemia (FA) pathway genes, chromosome maintenance genes and others. The current study focused on exploiting the conditional vulnerabilities caused by TTFields exposure to test novel combination therapies that target DNA replication fork stability combined with radiation. <h3>Materials/Methods</h3> Human NSCLC cell lines (H157, H4006, H549 and H1299) and pancreatic cancer cell lines (PANC-1 and Panc 04.03) were used in this study. A laboratory research system was used to generate TTFields. TTFields, radiation and drug combination efficacy were tested using clonogenic cell survival assays while the Highest Single Agent approach was used to assess the combinatorial efficacy. <h3>Results</h3> Comparative analysis of genomics and proteomics data revealed dysregulation of pathways including cell cycle, DNA damage repair and replication, and transcriptional and translational regulation under TTFields exposure. Functional characterization studies confirmed the downregulation of FA pathway proteins there by increased replication stress, DNA damage and impaired DNA damage repair. To exploit the conditional vulnerability environment induced by TTFields exposure different chemo agents that target and increase replication stress were tested in novel combination therapy options. It was found that the effect of TTFields exposure concomitant with the PARP1 inhibitor olaparib followed by irradiation (IR) was synergistic compared to IR or olaparib alone or in combination. In addition, TTFields synergistically enhanced the cell killing efficacy of the ATR inhibitor AZD6738 followed by IR. <h3>Conclusion</h3> Expression of FA pathway genes/proteins is modulated by TTFields exposure and likely explains the reduced DNA repair capacity and replication fork maintenance. Hence, targeting DNA damage repair and the DNA replication stress pathway using inhibitors of PARP1 and ATR could be used in combination with TTFields with or without radiation for cancer therapy.

MTA2 sensitizes gastric cancer cells to PARP inhibition by induction of DNA replication stress

Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib selectively kills cancer cells with BRCA-deficiency and is approved for BRCA-mutated breast, ovarian and pancreatic cancers by FDA. However, phase III study of olaparib failed to show a significant improvement in overall survival in patients with gastric cancer (GC). To discover an effective biomarker for GC patient-selection in olaparib treatment, we analyzed proteomic profiling of 12 GC cell lines. MTA2 was identified to confer sensitivity to olaparib by aggravating olaparib-induced replication stress in cancer cells. Mechanistically, we applied Cleavage Under Targets and Tagmentation assay to find that MTA2 proteins preferentially bind regions of replication origin-associated DNA sequences, which could be enhanced by olaparib treatment. Furthermore, MTA2 was validated here to render cancer cells susceptible to combination of olaparib with ATR inhibitor AZD6738. In general, our study identified MTA2 as a potential biomarker for olaparib sensitivity by aggravating olaparib-induced replication stress.

The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

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.

Abstract 5427: Genomic prevalence analysis in TNBC patients in the Phase 2 VIOLETTE study

Abstract Introduction: Triple-negative breast cancer (TNBC) accounts for ~15-20% of all breast carcinomas. VIOLETTE is a global, multicentre, open-label, randomized phase 2b study (NCT03330847), recruiting patients with metastatic TNBC to 2 treatment arms: olaparib and olaparib + ceralasertib (ATR inhibitor AZD6738). Patients are prospectively tested for mutations in genes involved in homologous recombination repair (HRR) by tumor DNA sequencing for stratification into BRCA mutated (BRCAm), non-BRCA HRR mutated (HRRm), and HRR wild-type (HRRwt) strata in each treatment arm. Patients can also be recruited as a result of a local test and tumor sample for those patients is analysed retrospectively. Methods: Sequencing of VIOLETTE tumor samples (prospective and retrospective) (n=518; DCO Sep 2019) using the FoundationOneCDx Clinical trial assay (CTA) test (Foundation Medicine [FMI]) was conducted to detect deleterious/suspected deleterious alterations, classified using AstraZeneca rules. Mutational zygosity and germline vs somatic status were assessed by a Somatic/ Germline/ Zygosity (SGZ) algorithm at FMI (Sun et al., 2018) and genome-wide LOH (loss of heterozygosity - gLOH) scores were generated. Results: The most prevalent alterations were in TP53 (86%) and MYC (25%). Alterations in main cancer signaling pathways included the HRR pathway (23%: BRCA1, BRCA2, ATM, RAD51B, RAD51C, RAD54L, RAD51D, BRIP1, FANCL, PALB2, BARD1, CHEK1, CHEK2, CDK12, PPP2R2A), the PI3K pathway (39%; (PIK3CA - 17.8%; PTEN - 20.1%; AKT1 - 5%)), cell cycle (37%: RB1, CDKN2A, CCNE1, CCND1) and RTK/RAS (22%: NF1, KRAS, FGFR1, FGF19/FGF3/FGF4). Preclinical and clinical data suggest that alterations in HRR pathway genes are associated with sensitivity to PARP inhibitors including olaparib. The most prevalent HRR gene alterations were BRCA1 (9.5%) and BRCA2 (3%) and non-BRCAm HRRm prevalence was 11%. Out of 96 patients with reported zygosity, 81% (44/54) of BRCAm and 64% (27/42) of non-BRCAm HRRm tumors had biallelic inactivation. Consistent with gLOH being a measure of HRR deficiency, gLOH scores were significantly higher in BRCAm patients compared to HRRwt patients (mean BRCAm 28, mean HRRwt 19, P=2.1E-08). Interestingly, we observed a significant mutual exclusivity between alterations in the HRR pathway and activating PIK3CA variants (P=0.01), suggesting distinct disease segments within TNBC. Conclusions: The genomic prevalence of breast cancer related alterations in VIOLETTE is as expected for TNBC, excepting HRRm, which was lower than anticipated from the TCGA dataset (23% VIOLETTE vs 35% TCGA). This may reflect the specific eligibility criteria for VIOLETTE. This is the largest dataset of genomic prevalence in TNBC reported, providing a deeper understanding of the prevalence of potentially actionable alterations to guide clinical development of targeted therapies. Citation Format: Joshua Armenia, Zhongwu Lai, Andrew Tutt, Emma Dean, Bienvenu Loembe, Hannah McGarvey, Sarah Edgington, J Carl Barrett, Andrew J. Pierce, Elizabeth A. Harrington, Natalia Lukashchuk. Genomic prevalence analysis in TNBC patients in the Phase 2 VIOLETTE study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5427.

Differential therapeutic effects of PARP and ATR inhibition combined with radiotherapy in the treatment of subcutaneous versus orthotopic lung tumour models

BackgroundSubcutaneous mouse tumour models are widely used for the screening of novel antitumour treatments, although these models are poor surrogate models of human cancers.MethodsWe compared the antitumour efficacy of the combination of ionising radiation (IR) with two DNA damage response inhibitors, the PARP inhibitor olaparib and the ATR inhibitor AZD6738 (ceralasertib), in subcutaneous versus orthotopic cancer models.ResultsOlaparib delayed the growth of irradiated Lewis lung carcinoma (LL2) subcutaneous tumours, in agreement with previous reports in human cell lines. However, the olaparib plus IR combination showed a very narrow therapeutic window against LL2 lung orthotopic tumours, with nearly no additional antitumour effect compared with that of IR alone, and tolerability issues emerged at high doses. The addition of AZD6738 greatly enhanced the efficacy of the olaparib plus IR combination treatment against subcutaneous but not orthotopic LL2 tumours. Moreover, olaparib plus AZD6738 administration concomitant with IR even worsened the response to radiation of head and neck orthotopic tumours and induced mucositis.ConclusionsThese major differences in the responses to treatments between subcutaneous and orthotopic models highlight the importance of using more pathologically relevant models, such as syngeneic orthotopic models, to determine the most appropriate therapeutic approaches for translation to the clinic.

Abstract PR08: Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC

Abstract Background: Standard treatment for head and neck squamous cell carcinoma (HNSCC) includes platinum-based therapy given in combination with radiation. Although platinum has been a key drug of HNSCC treatment for decades, many patients develop resistance and the prognosis is unsatisfactory, and therefore development of novel treatment strategies is needed. Targeting ATR has been shown to be effective as a monotherapy in various type of cancers. Thus, inhibition of ATR could be a new approach when combined with DNA damaging agents to kill cancer cells. Here, we hypothesize that addition of ATR inhibitor AZD6738 can enhance the antitumor effects of cisplatin in HNSCC cells and result in better survival in preclinical models of HNSCC. Materials and Methods: Clonogenic survival assays, reverse phase protein array (RPPA), Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the in vitro sensitivity of HNSCC cells to AZD6738 and cisplatin and the molecular mechanisms associated with antitumor response. An orthotopic mouse model of oral cancer was used to explore in vivo efficacy of the combination treatment. Results: HNSCC cells treated with various concentrations of AZD6738 showed an IC50 value of 0.24-3.00 μmol/L and no differential sensitivity was observed among cells with different TP53 mutational status. Treatment with constant drug concentration ratios of AZD6738 and cisplatin (2:1) resulted in a combination index of &amp;lt;1, indicating synergistic effects. RPPA pointed out proteins involved in DNA repair, DNA replication, and cell cycle to be predominantly changed in the combination groups compared to single treatments. Among these, the replication factors Cdc6 and Cdt1 were both reduced with the combination treatment. Furthermore, Western blot results revealed that combination treatments lead to enhanced DNA damage indicated by increased phosphorylation of H2AX and induction of replication stress marker (decrease of phospho-CDK1 and phospho-CHK1). Moreover, levels of RRM2 and Rad51 were diminished, suggesting insufficient nucleotide supply and impaired homologous replication. Combining both drugs also resulted in a time-dependent increase of cleaved PARP. This apoptotic death was further confirmed by Annexin V/PI assay. Cell cycle analysis and live cell imaging revealed that an accumulation of cells in the S phase was more than doubled after 12 and 48 hours, indicating replicative stress. In a pilot in vivo study, cisplatin combined with AZD6738 reduced tumor growth in mice bearing oral tumors. Conclusions: Addition of AZD6738 synergizes with cisplatin in vitro and potentiates cisplatin response in vivo. Mechanistically, combination of cisplatin and AZD6738 induces DNA damage and replication stress, resulting in apoptotic cell death. Interestingly, proteins essential for the origin licensing as part of the DNA replication initiation machinery were decreased following combination treatment, offering a potential mechanistic insight and warranting further investigation. Citation Format: Antje Lindemann, Hideaki Takahashi, Ameeta A. Patel, Walter N. Hittelman, Abdullah A. Osman, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR08.

Combined PARP and ATR inhibition potentiates genome instability and cell death in ATM-deficient cancer cells

The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1–2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib.

ATR inhibitor AZD6738 enhances the antitumor activity of radiotherapy and immune checkpoint inhibitors by potentiating the tumor immune microenvironment in hepatocellular carcinoma

BackgroundRadioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation...