AZD1390, an ataxia telangiectasia mutated inhibitor, attenuates microglia-mediated neuroinflammation and ischemic brain injury.

AIMS: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown. METHODS: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay. RESULTS: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway. CONCLUSION: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke.

Treatment for ovarian clear cell carcinoma with combined inhibition of WEE1 and ATR.

BACKGROUND: Standard platinum-based therapy for ovarian cancer is inefficient against ovarian clear cell carcinoma (OCCC). OCCC is a distinct subtype of epithelial ovarian cancer. OCCC constitutes 25% of ovarian cancers in East Asia (Japan, Korea, China, Singapore) and 6-10% in Europe and North America. The cancer is characterized by frequent inactivation of ARID1A and 10% of cases of endometriosis progression to OCCC. The aim of this study was to identify drugs that are either FDA-approved or in clinical trials for the treatment of OCCC. RESULTS: High throughput screening of 166 compounds that are either FDA-approved, in clinical trials or are in pre-clinical studies identified several cytotoxic compounds against OCCC. ARID1A knockdown cells were more sensitive to inhibitors of either mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738) or MDM2 (RG7388) compared to control cells. Also, compounds targeting BH3 domain (AZD4320) and SRC (AZD0530) displayed preferential cytotoxicity against ARID1A mutant cell lines. In addition, WEE1 inhibitor (AZD1775) showed broad cytotoxicity toward OCCC cell lines, irrespective of ARID1A status. CONCLUSIONS: In a selection of 166 compounds we showed that inhibitors of ATR and WEE1 were cytotoxic against a panel of OCCC cell lines. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC.

Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer.

BACKGROUND: Targeting the DNA damage repair (DDR) pathways is an attractive strategy for boosting cancer immunotherapy. Ceralasertib (AZD6738) is an oral kinase inhibitor of ataxia telangiectasia and Rad3 related protein, which is a master regulator of DDR. We conducted a phase II trial of ceralasertib plus durvalumab in patients with previously treated advanced gastric cancer (AGC) to demonstrate the safety, tolerability, and clinical activity of the combination. METHODS: This phase II, open-label, single-center, non-randomized study was designed to evaluate the efficacy and safety of ceralasertib in combination with durvalumab in patients with AGC. The study drug regimen was ceralasertib (240 mg two times a day) days 15-28 in a 28-day cycle in combination with durvalumab (1500 mg) at day 1 every 4 weeks. The primary end point was overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors (V.1.1). Exploratory biomarker analysis was performed using fresh tumor biopsies in all enrolled patients. RESULTS: Among 31 patients, the ORR, disease control rate, median progression-free survival (PFS), and overall survival were 22.6% (95% CI 9.6% to 41.1%), 58.1% (95% CI 39.1% to 75.5%), 3.0 (95% CI 2.1 to 3.9) months, and 6.7 (95% CI 3.8 to 9.6) months, respectively. Common adverse events were manageable with dose modification. A subgroup of patients with a loss of ataxia telangiectasia mutated (ATM) expression and/or high proportion of mutational signature attributable to homologous repair deficiency (sig. HRD) demonstrated a significantly longer PFS than those with intact ATM and low sig. HRD (5.60 vs 1.65 months; HR 0.13, 95% CI 0.045 to 0.39; long-rank p<0.001). During the study treatment, upregulation of the innate immune response by cytosolic DNA, activation of intratumoral lymphocytes, and expansion of circulating tumor-reactive CD8 +T cell clones were identified in responders. Enrichment of the tumor vasculature signature was associated with treatment resistance. CONCLUSIONS: Ceralasertib plus durvalumab has promising antitumor activity, with durable responses in patients with refractory AGC. Thus, a biomarker-driven trial is required. TRIAL REGISTRATION: NCT03780608.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer.

BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors. RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

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

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

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.

We determine that type I interferon (IFN) response biomarkers are enriched in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors; however, actionable vulnerabilities associated with IFN signaling have not been systematically defined. Integration of a phosphoproteomic analysis and a chemical genomics synergy screen reveals that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) in PDAC cells and sensitizes them to ATR inhibitors. IFN triggers cell-cycle arrest in S-phase, which is accompanied by nucleotide pool insufficiency and nucleoside efflux. In combination with IFN, ATR inhibitors induce lethal DNA damage and downregulate nucleotide biosynthesis. ATR inhibition limits the growth of PDAC tumors in which IFN signaling is driven by stimulator of interferon genes (STING). These results identify a cross talk between IFN, DNA replication stress response networks, and nucleotide metabolism while providing the rationale for targeted therapeutic interventions that leverage IFN signaling in tumors.

Discovery of small-molecule ATR inhibitors for potential cancer treatment: a patent review from 2014 to present.

INTRODUCTION: Ataxia telangiectasia and RAD3-related kinase (ATR) is one of the key phosphatidylinositol 3-kinase-related kinase family members important for DNA damage response and repair pathways. Targeting ATR kinase for potential cancer therapy has attracted a great deal of attention to both pharmaceutical industries and academic community. AREA COVERED: This article surveys the patents published since 2014 aiming to analyze the structural features of scaffolds and the patent space. It also discusses the recent clinical developments and provides perspectives on the challenges and the future directions. EXPERT OPINION: ATR kinase appears to be a viable drug target for anticancer therapy. Similar to DNA-PK inhibitors, the clinical investigation of an ATRi employs both monotherapy and combination strategy. In the combination strategy, an ATRi is typically combined with a radiation or a targeted drug such as chemotherapy agent poly (ADP-ribose) polymerase (PARP) inhibitor, etc. Diverse structures comprising different scaffolds from mono-heteroaryl to bicyclic heteroaryl to tricyclic heteroaryl to macrocycle are capable of achieving good ATR inhibitory activity and good ATR selectivity over other closely related enzymes. There are eight ATR inhibitors currently being evaluated in clinics, with the hope to get approval in the near future.

Primary Founder Mutations in the PRKDC Gene Increase Tumor Mutation Load in Colorectal Cancer.

The clonal composition of a malignant tumor strongly depends on cellular dynamics influenced by the asynchronized loss of DNA repair mechanisms. Here, our aim was to identify founder mutations leading to subsequent boosts in mutation load. The overall mutation burden in 591 colorectal cancer tumors was analyzed, including the mutation status of DNA-repair genes. The number of mutations was first determined across all patients and the proportion of genes having mutation in each percentile was ranked. Early mutations in DNA repair genes preceding a mutational expansion were designated as founder mutations. Survival analysis for gene expression was performed using microarray data with available relapse-free survival. Of the 180 genes involved in DNA repair, the top five founder mutations were in PRKDC (n = 31), ATM (n = 26), POLE (n = 18), SRCAP (n = 18), and BRCA2 (n = 15). PRKDC expression was 6.4-fold higher in tumors compared to normal samples, and higher expression led to longer relapse-free survival in 1211 patients (HR = 0.72, p = 4.4 × 10-3). In an experimental setting, the mutational load resulting from UV radiation combined with inhibition of PRKDC was analyzed. Upon treatments, the mutational load exposed a significant two-fold increase. Our results suggest PRKDC as a new key gene driving tumor heterogeneity.

Dose-dependent bioavailability and tissue distribution of the ATR inhibitor AZD6738 (ceralasertib) in mice.

PURPOSE: Ataxia telangiectasia and Rad3-related (ATR) initiates and regulates cellular responses to DNA damage, such as those caused by cancer treatments. Several ATR inhibitors (ATRi) are in clinical development including AZD6738. Therapeutic indices among ATRi may differ as a result of varying potencies and concentrations at both tumor and off-target sites. Additionally, AZD6738 contributes to anti-tumor immune responses necessitating evaluation of exposure at immunological sites. METHODS: Using mouse models and a highly sensitive LC-MS/MS assay, the pharmacokinetics of AZD6738 were studied, including dose linearity, bioavailability, metabolism, and tissue distribution in tumor-bearing mice. RESULTS: Initial studies identified dose-dependent bioavailability, with greater than proportional increases in exposure as dose increased resulting in a ~ twofold increase in bioavailability between the lowest and highest investigated doses. These behaviors were successfully captured with a compartmental PK model. Analysis of metabolite PK revealed decreasing metabolic ratios with increasing dose, indicative of saturable first-pass metabolism. Further analysis revealed that intestinal and gut metabolism contribute to metabolism and these saturable mechanisms. Studies of tumor and tissue distribution found rapid and extensive drug distribution to most tissues except brain and spinal cord. CONCLUSION: The complex non-linear behavior of AZD6738 PK in mice was due to pre-systemic saturation and which appears to be recapitulated clinically at low doses. PK reported here will allow future correlation of tissue related toxicities with drug exposure as well as exposure with immunological responses. These results can also be compared with those from similar studies of other ATRi to contrast drug exposure with responses.

RP-3500: A Novel, Potent, and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.

Inhibition of triple­negative breast cancer proliferation and motility by reactivating p53 and inhibiting overactivated Akt.

Mutations of p53 tumor suppressors occur more frequently in cancers at advanced stages or in more malignant cancer subtypes such as triple­negative breast cancer. Thus, restoration of p53 tumor suppressor function constitutes a valuable cancer therapeutic strategy. In the present study, it was revealed that a specific inhibitor of histone deacetylase 6, ACY­1215, caused increased acetylation of p53 in breast cancer cells with mutated p53, which was accompanied by increased expression of p21. These results suggested that ACY­1215 may lead to enhanced transcriptional activity of p53. It was also determined that ACY­1215 treatment resulted in G1 cell cycle arrest and apoptosis in these cancer cells. Furthermore, ACY­1215 displayed a synergistic effect with specific inhibitors of ATM, an activator of Akt, in inducing cancer cell apoptosis and inhibiting their motility. More importantly, it was observed that combination of ACY­1215 and ATM inhibitors exhibited markedly more potent antitumor activity than the individual compound in xenograft mouse models of breast cancer with mutant p53. Collectively, our results demonstrated that ACY­1215 is a novel chemotherapeutic agent that could restore mutant p53 function in cancer cells with strong antitumor activity, either alone or in combination with inhibitors of the ATM protein kinase.

New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors.

The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

DNA Double-strand Break Signaling Is a Therapeutic Target in Head and Neck Cancer.

BACKGROUND: Head and neck cancer (HNC) is common worldwide. Given poor outcomes for patients with HNC, research into targeted therapies is needed. Ataxia telangiectasia mutated (ATM) is a DNA damage kinase which is activated by double-strand DNA breaks. We tested the effects of a novel ATM inhibitor on HNC cell lines and xenografts. MATERIALS AND METHODS: p53-Binding protein 1 and phosphorylated ATM were localized in cultured cells by immunofluorescence microscopy. Protein expression was determined by western blot. Tumor xenografts were established by injecting HNC lines into immunocompromised mice. Tumor sections were characterized by immunohistochemistry. Apoptotic cells were determined by terminal transferase-mediated dUTP nick-end labeling assay. RESULTS: ATM inhibition increased double-strand DNA breaks at replication foci in HNC cell lines. ATM inhibition affected cell-cycle regulatory protein expression, blocked cell-cycle progression at the G2/M phase and resulted in apoptosis. CONCLUSION: ATM inhibition may be therapeutically useful in treating HNC.

ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.

CCNE1-amplified ovarian cancers (OVCAs) and endometrial cancers (EMCAs) are associated with platinum resistance and poor survival, representing a clinically unmet need. We hypothesized that dysregulated cell-cycle progression promoted by CCNE1 overexpression would lead to increased sensitivity to low-dose WEE1 inhibition and ataxia telangiectasia and Rad3-related (ATR) inhibition (WEE1i-ATRi), thereby optimizing efficacy and tolerability. The addition of ATRi to WEE1i is required to block feedback activation of ATR signaling mediated by WEE1i. Low-dose WEE1i-ATRi synergistically decreases viability and colony formation and increases replication fork collapse and double-strand breaks (DSBs) in a CCNE1 copy number (CN)-dependent manner. Only upon CCNE1 induction does WEE1i perturb DNA synthesis at S-phase entry, and addition of ATRi increases DSBs during DNA synthesis. Inherent resistance to WEE1i is overcome with WEE1i-ATRi, with notable durable tumor regressions and improved survival in patient-derived xenograft (PDX) models in a CCNE1-level-dependent manner. These studies demonstrate that CCNE1 CN is a clinically tractable biomarker predicting responsiveness to low-dose WEE1i-ATRi for aggressive subsets of OVCAs/EMCAs.

Combination ATR and PARP Inhibitor (CAPRI): A phase 2 study of ceralasertib plus olaparib in patients with recurrent, platinum-resistant epithelial ovarian cancer.

OBJECTIVE: Platinum-resistant, high-grade serous ovarian cancer (HGSOC) has limited treatment options. Preclinical data suggest that poly(ADP-ribose) polymerase inhibitors (PARPi) and ataxia telangiectasia and Rad3-related kinase inhibitors (ATRi) are synergistic. CAPRI (NCT03462342) is an investigator-initiated study of olaparib plus ceralasertib in recurrent HGSOC. Herein, we present results from the platinum-resistant cohort. METHODS: A Simon 2-stage design was utilized. Platinum-resistant HGSOC patients received ceralasertib 160 mg orally daily, days 1-7 and olaparib 300 mg orally twice daily, days 1-28 of a 28-day cycle until toxicity or progression. Primary endpoints were toxicity and efficacy including objective response rate (ORR) by RECIST. Secondary endpoint was progression-free survival (PFS). The null hypothesis (≤5% ORR) would be rejected if there were ≥ 1 responses in 12 patients. RESULTS: Fourteen PARPi-naïve patients were evaluable for toxicity; 12 were evaluable for response. Three had BRCA1 mutations (1 germline, 2 somatic). Adverse events possibly related to treatment were primarily grade (G) 1/2. G3 toxicities included nausea (14.3%), fatigue (7.1%), anorexia (7.1%), and anemia (7.1%). No objective responses occurred. Best response was stable disease in 9 patients and progressive disease in three. Five patients had a ≥ 20% to <30% reduction in disease burden, including 3 with BRCA1 mutations. Three of 11 patients (27%; 2 with BRCA1 mutations) evaluable by Gynecologic Cancer Intergroup criteria had >50% CA-125 decline, including 2 with CA-125 normalization. Median PFS was 4.2 months overall (90% CI:3.5-8.2) and 8.2 months (3.6 months-not determined) for patients with BRCA1 mutations. CONCLUSIONS: Olaparib plus ceralasertib is well-tolerated. No objective responses occurred, though a signal of activity was seen particularly in disease associated with BRCA1. Further evaluation of this combination should include alternate dosing strategies in genomically-selected populations.

A Replication stress biomarker is associated with response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in ovarian cancer.

In a trial of patients with high grade serous ovarian cancer (HGSOC), addition of the ATR inhibitor berzosertib to gemcitabine improved progression free survival (PFS) compared to gemcitabine alone but biomarkers predictive of treatment are lacking. Here we report a candidate biomarker of response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in HGSOC ovarian cancer. Patients with replication stress (RS)-high tumors (n = 27), defined as harboring at least one genomic RS alteration related to loss of RB pathway regulation and/or oncogene-induced replication stress achieve significantly prolonged PFS (HR = 0.38, 90% CI, 0.17-0.86) on gemcitabine monotherapy compared to those with tumors without such alterations (defined as RS-low, n = 30). However, addition of berzosertib to gemcitabine benefits only patients with RS-low tumors (gemcitabine/berzosertib HR 0.34, 90% CI, 0.13-0.86) and not patients with RS-high tumors (HR 1.11, 90% CI, 0.47-2.62). Our findings support the notion that the exacerbation of RS by gemcitabine monotherapy is adequate for lethality in RS-high tumors. Conversely, for RS-low tumors addition of berzosertib-mediated ATR inhibition to gemcitabine is necessary for lethality to occur. Independent prospective validation of this biomarker is required.

Ceralasertib-Mediated ATR Inhibition Combined With Olaparib in Advanced Cancers Harboring DNA Damage Response and Repair Alterations (Olaparib Combinations).

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapy in cancers with homologous recombination repair deficiency. However, efficacy is limited by both intrinsic and acquired resistance. The Olaparib Combinations basket trial explored olaparib alone and in combination with other homologous recombination-directed targeted therapies. Here, we report the results of the arm in which olaparib was combined with the orally bioavailable ataxia telangiectasia and RAD3-related inhibitor ceralasertib in patients with relapsed or refractory cancers harboring DNA damage response and repair alterations, including patients with BRCA-mutated PARP inhibitor-resistant high-grade serous ovarian cancer (HGSOC). PATIENTS AND METHODS: Germline and somatic mutations had to be deleterious by COSMIC or ClinVar for eligibility. Olaparib was administered at 300 mg twice daily and ceralasertib at 160 mg daily on days 1-7 in 28-day cycles until progression or unacceptable toxicities. Primary end points were confirmed complete response (CR) or partial response (PR) rates and clinical benefit rate (CBR; CR + PR + stable disease [SD] at 16 weeks). RESULTS: Twenty-five patients were enrolled, with median four prior therapies. Five patients required dose reductions for myelosuppression. Overall response rate was 8.3% and CBR was 62.5% among the entire cohort. Two of five patients with tumor harboring ATM mutation achieved CR or SD ongoing at 24+ months, respectively (CBR 40%). Of seven patients with PARP inhibitor-resistant HGSOC, one achieved PR (-90%) and five had SD ranging 16-72 weeks (CBR 86%). CONCLUSION: Olaparib with ceralasertib demonstrated preliminary activity in ATM-mutated tumors and in PARP inhibitor-resistant BRCA1/2-mutated HGSOC. These data warrant additional studies to further confirm activity in these settings.

Molecular basis of human ATM kinase inhibition.

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.