Rad3-related Protein Kinase Research Articles

A phase I study of highly potent oral ATR inhibitor (ATRi) tuvusertib plus oral PARP inhibitor (PARPi) niraparib in patients with solid tumors.

3018 Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase and poly-ADP ribose polymerases (PARPs) are crucial components in the DNA damage response (DDR). Combining tuvusertib and niraparib may synergistically enhance synthetic lethality and increase apoptosis. Part B1 of the DDRiver Solid Tumors 301 study (NCT04170153) assessed this combination. Methods: Part B1 of this open-label multicenter dose-escalation phase Ib trial enrolled unselected patients with metastatic or locally advanced unresectable solid tumors refractory to standard treatment. The primary objective was to determine safety (including maximum tolerable dose and recommended dose(s) for expansion [RDE]). Secondary and tertiary objectives included determination of the pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of different dosing regimens of tuvusertib plus niraparib. Multiple continuous and intermittent schedules were explored with tuvusertib doses ranging from 90–180 mg once-daily (QD) and niraparib doses from 100–200 mg QD. A partial ordering continual reassessment method was used to identify the RDEs. Results: As of 02 January 2024, 43 patients had been enrolled (all with baseline body weight <77kg or platelet count <150,000/mm3); 13 patients remained on treatment. Ten (26.3%) patients had dose-limiting toxicities (DLTs). The most common DLT was grade 3 anemia requiring blood transfusion (n=4; 10.5%); the most frequent grade ≥3 treatment-emergent adverse events were anemia (n=18; 41.9%), platelet count decrease (n=6; 14.0%) and fatigue (n=4; 9.3%). The PK of tuvusertib when combined with niraparib was consistent with that of tuvusertib monotherapy, suggesting a lack of any clinically meaningful mutual drug–drug interactions. Dose-dependent ɣ-H2AX inhibition (proximal PD marker) was observed at tuvusertib doses ≥130 mg QD. Preliminary efficacy data show 5 (15.6%) responses (3 confirmed) by RECIST v1.1 in 32 evaluable patients: 2 in patients with epithelial ovarian cancer (EOC; 1 with BRCA1-mutant [ BRCA1m] PARPi-resistant EOC, 1 with BRCA-wild type homologous recombination deficiency-positive EOC) and 1 each in patients with non-small cell lung cancer, estrogen receptor-positive HER2-negative BRCA1m breast cancer, and BRCA1m PARPi-resistant pancreatic cancer. Tuvusertib 180 mg QD and niraparib 100 mg QD or tuvusertib 90 mg QD and niraparib 200 mg QD, both given in a 1 week on/1 week off schedule, were identified as RDEs. Conclusions: The combination of tuvusertib plus niraparib, each administered on a 1 week on/1 week off schedule, has a manageable safety profile and is suitable for further investigation. A combination study in patients with PARPi-resistant EOC is planned. Clinical trial information: NCT04170153 .

A phase I study of ATR inhibitor BAY1895344 (elimusertib) plus topotecan (ETCTN 10402): Results of dose escalation.

3076 Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is activated by replication stress and recruited to stalled forks or single strand DNA abnormalities in various cancers. The topoisomerase-I (Top1) inhibitor topotecan induces DNA damage. The ATR inhibitor BAY 1895344 (elimusertib) has demonstrated cytotoxic potential in small cell lung cancer and gastrointestinal cancer xenografts when combined with Top1 inhibitors . Methods: This is phase Ia study of elimusertib combined with topotecan in adult patients with refractory advanced solid tumors for whom topotecan can be considered as part of standard care. Patients with previous topotecan exposure were excluded. The study combination was assessed starting at Topotecan 1 mg/m2 IV (D1-D5) plus elimusertib 20 mg BID (D2, D5) (Cycle=21 days). Dose escalation utilized a 3+3 design. Primary objectives were to assess safety and tolerability and estimate the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of the combination. Secondary objectives included estimating pharmacokinetic (PK) profiles and assessing anti-tumor activity of the combinations. Results: Eight patients were treated in dose escalation. Participants were a median of 63 years old and had received 2 median lines of previous therapy. The initial two patients enrolled into dose level (DL) 1 had dose-limiting toxicities with one of patient experiencing respiratory failure and cardiac arrest in setting of pancytopenia related to study drug. Six patients were subsequently enrolled in and received DL-1 (elimusertib decreased from 20 mg BID to 20 mg Daily) and no DLTs were observed. Notable grade 3+ treatment-related adverse events and summary best response are summarized in Table. Disease control rate was 43% among evaluation patient including one unconfirmed partial response. Median progression-free survival in the RP2D cohort was 3.45 months. PK studies are in process and results will be presented at time of meeting. Conclusions: RP2D and MTD was established for elimusertib in combination with Topotecan: Topotecan 1 mg/m2 IV [D1-D5] plus elimusertib 20 mg Daily [D2, D5] every 3 weeks. Dose escalation was notably limited by myelotoxicity. Due to sponsor decision, the study was halted prior to planned expansions cohorts but the concept of ATR + topo I inhibition remains relevant. Clinical trial information: NCT04514497 . [Table: see text]

Pharmacokinetic (PK) and pharmacodynamic (PD) findings from a phase 1b study of ATR inhibitor tuvusertib + anti-PD-L1 avelumab in patients with advanced unresectable solid tumors.

2614 Background: Ataxia telangiectasia and Rad3-related protein (ATR) kinase is critical in the DNA damage response, and its inhibition modulates antitumor immunity. The combination of ATR inhibitor (ATRi) + immune checkpoint inhibitor (ICI) has shown activity in patients with ICI-resistant tumors and may have the potential to overcome ICI resistance and induce antitumor immune responses. Methods: Part B1 of the open-label, multicenter study DDRiver Solid Tumors 320 investigated safety, tolerability, PK, and PD, including effects on immune cells, of ATRi tuvusertib in combination with the ICI avelumab (anti-PD-L1) in patients with advanced unresectable solid tumors. Flow cytometry was used to analyze tuvusertib target inhibition via γ-H2AX modulation in the CD45+ fraction of ex-vivo stimulated peripheral blood mononuclear cells, and to explore the effect on the peripheral immunophenotype. Tuvusertib PK samples were analyzed by a validated bioanalytical liquid chromatography/mass spectrometry method. Results: 22 patients were enrolled and treated with tuvusertib 180 mg once daily on a schedule of 2 weeks (w) on treatment followed by a treatment break of 1 or 2 w, and avelumab 800 mg once every 2 weeks (Q2W). The 2 w on/1 w off schedule, corresponding to the recommended dose for expansion (RDE) for tuvusertib monotherapy, was chosen for further exploration. At this schedule, 2 of 9 patients evaluable for dose-limiting toxicity (DLT) experienced DLTs: Grade 3 ALT and Grade 3 AST increase (n=1), and Grade 3 anemia requiring transfusion (n=1). A patient with chordoma experienced a RECIST v1.1 partial response. Preliminary PK data for tuvusertib suggested rapid absorption with median Tmax range of ~2–3 h and mean elimination half-life range of ~2.93 to 4.23 h, with ~2-fold accumulation of steady-state area-under-the-curve following multiple doses. Exposure of tuvusertib in combination with avelumab was consistent with tuvusertib monotherapy exposure (1). PD showed complete or almost complete target inhibition at 1–6 h after tuvusertib 180 mg followed by rebound above baseline after 24 h on days 1 and 8 of cycle 1. No clear trend of variation in absolute counts of myeloid-derived suppressor cells, T and B lymphocytes, monocytes, and natural killer cell subtypes was detected. Conclusions: Tuvusertib and avelumab were combined at established monotherapy doses with no new safety findings. Tuvusertib PD and exposure data were in line with monotherapy observations. The combination did not cause any consistent change of the immunophenotype. Given the accumulating evidence of ATRi as an immunosensitiser (2), further evaluation of this combination in patients with ICI-resistant advanced solid tumors is warranted. 1. Yap T et al., Ann Oncol 2022;33(suppl_7):S197–S224. 2. Besse B et al., JTO 2022;17(suppl_9):S41–S42. Clinical trial information: NCT05396833 .

Pharmacodynamic (PD) and immunophenotyping analyses of ATR inhibitor (ATRi) tuvusertib + ATM inhibitor (ATMi) lartesertib in a phase Ib study in patients with advanced unresectable solid tumors.

2612 Background: Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) protein kinases orchestrate the DNA damage response. A synthetic lethal relationship between ATR and ATM genes in cancer has been described1 and ATMi synergistically potentiate the efficacy of ATRi in vitro and in vivo.2 The combination of tuvusertib + lartesertib was investigated in Part A1 of the open-label, multicenter study DDRiver Solid Tumors 320 in patients with advanced unresectable solid tumors. Here, we report results of the PD and immunophenotyping analyses. Methods: PD analysis comprised γ-H2AX in serial blood samples, stimulated ex vivowith 4-NQO, bleomycin, or controls. Flow cytometry was used to measure target inhibition via γ-H2AX modulation in the CD45+ lymphocytes fraction and to explore the effect of tuvusertib + lartesertib on the immunophenotype (myeloid-derived suppressor cells, T and B lymphocytes, monocytes, and natural killer cells subsets) in serial blood samples. Pharmacokinetic samples were analyzed by a validated bioanalytical liquid chromatography/mass spectrometry method. Time-matched blood samples were collected at baseline, 1, 3, 6, and 24 hours after first tuvusertib and lartesertib administration on days 1 and 8 of cycle 1 for the γ-H2AX analysis, and on days 1 and 15 of cycles 1 and 2 before treatment for immunophenotyping. Results: Immunophenotyping data and γ-H2AX levels were obtained from 41 and 34 patients, respectively. For tuvusertib, complete or almost complete target inhibition was seen at 1–6 hours after treatment, followed by a rebound above baseline after 24 hours, on both days 1 and 8 at doses of 130 and 180 mg once daily (QD). For lartesertib, a variable target inhibition of approximately 50 % on average was seen across all time points at doses of 100, 150 and 200 mg QD. No target inhibition was seen for tuvusertib at 90 mg QD and lartesertib at 50 mg QD (cohort 1). Tuvusertib + lartesertib induced a transient decrease of monocytes and natural killer (NK) cells, with partial or complete recovery to baseline levels during treatment breaks in schedules of 2 weeks on treatment followed by a treatment break of 1 or 2 weeks, respectively. Conclusions: Tuvusertib and lartesertib combination PD outcomes were in line with respective monotherapy observations.3,4 The combination did not cause any consistent change in the levels of immune cell subsets at all dose levels tested, except a mild, transient decrease in monocytes and NK cells, in line with the tuvusertib monotherapy observations. 1. Kantidze et al., Trends Cancer 2018;4(11):755–68. 2. Turchick et al., Mol Cancer Ther 2023;22(7):859–72. 3. Yap et al., Ann Oncol 2022;33(suppl_7):S197–S224. 4. Siu et al., Cancer Res 2023;83(8_Suppl):CT171. Clinical trial information: NCT05396833 .

Abstract CT063: Phase Ib trial of ATR inhibitor (ATRi) tuvusertib + ATM inhibitor (ATMi) lartesertib (M4076) in patients (pts) with advanced solid tumors

Abstract Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) protein kinases are critical in the DNA damage response. ATR and ATM genes have a synthetic lethal relationship in cancer and ATMi synergistically potentiates the efficacy of ATRi in vitro and in vivo. Here, we studied the combination potential of the highly potent, oral agents tuvusertib and lartesertib. The open-label, multicenter study DDRiver Solid Tumors 320 (NCT05396833) investigated safety, tolerability, PK and PD of tuvusertib + lartesertib in pts with advanced solid tumors refractory to standard therapy. PK samples were analyzed by a validated LC/MS method, and PD via γ-H2AX in ex vivo-stimulated CD45+ fraction by flow cytometry. As of 03.01.2024, 42 pts received ascending doses of tuvusertib + lartesertib. 5 pts experienced DLTs (Table 1). Frequent TEAEs (≥25 % of pts, any grade) were: anemia 62 %, nausea 45 %, fatigue and vomiting (43 % each). Grade ≥3 TEAEs occurred in 21 (50 %) pts; those in ≥3 pts were: anemia (n=9), decreased platelets (n=5), fatigue and decreased neutrophils (n=3 each). Preliminary steady-state PK showed rapid absorption: median Tmax of ~2-3 h (tuvusertib) and ~1-3.5 h (lartesertib); mean elimination half-life range of ~3.2-4.5 h (tuvusertib) and ~5.5-8.7 h (lartesertib); minimal accumulation. Exposure of the combination was consistent with respective monotherapies, indicating no clinically meaningful mutual drug-drug interactions. Preliminary PD showed complete or almost complete target inhibition 1-6 h after tuvusertib ≥130 mg and rebound above baseline after 24 h and target inhibition ~50 % across all time points at lartesertib ≥100 mg. 6 pts (2 ovarian, 2 uterine, 1 prostate cancer and 1 melanoma) had stable disease >16 weeks; 3 remain on treatment. Multiple tolerated combination doses were identified. Tuvusertib 180 mg QD + lartesertib 150 mg QD 2 w on/2 w off was selected for investigation in expansion cohorts in pts with prostate and endometrial cancer. Table 1. DLTs by ascending dose (DLT-evaluable patients) Dose (tuvusertib + lartesertib), mg N DLT DLT AE terms by cohort 2 w on/2 w off regimen 90 + 50 QD 3 No NA 130 + 50 QD 4 No NA 130 + 100 QD 3 No NA 180 + 100 QD 3 No NA 180 + 200 QD 4 Yes, in 2 pts One pt experienced febrile neutropenia Grade 3, neutrophil count decreased Grade 4, anemia Grade 3, platelet count decreased Grade 4, and candida infection Grade 2. The second pt had neutrophil count decreased Grade 4 180 + 150 QD 9 No NA 2 w on/1 w off regimen 180 + 150 QD 4 Yes, in 2 pts Both pts had platelet count decreased Grade 2 and Grade 3 180 + 100 QD 7 Yes, in 1 pt Neutrophil count decreased Grade 4 AE, adverse event; DLT, dose-limiting toxicities; N, DLT-evaluable patients; NA, not applicable; PD, pharmacodynamics; PCa, prostate cancer; PK, pharmacokinetics; pts, patients; QD, once daily, TEAE, treatment-emergent adverse event; w, week Citation Format: Lillian L. Siu, Elena Garralda Cabanas, Valentina Boni, Anthony W. Tolcher, Enrique Sanz Garcia, Jesús Fuentes-Antrás, Omar Saavedra, Deepthi S. Vagge, Giuseppe Locatelli, Burak Kürsad Günhan, Gregory Pennock, Jatinder Kaur Mukker, Ioannis Gounaris, Timothy A. Yap. Phase Ib trial of ATR inhibitor (ATRi) tuvusertib + ATM inhibitor (ATMi) lartesertib (M4076) in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT063.

Abstract 7171: Non-linear IV pharmacokinetics of the ATR inhibitor berzosertib (M6620) in mice

Abstract Introduction: The ataxia-telangiectasia and Rad3-related (ATR) protein kinase is an attractive target for cancer therapies as it is an apical initiator of DNA damage response (DDR) pathways that facilitate cell cycle arrest and DNA repair, thus promoting survival of cancer cells. Several ATR inhibitors (ATRi) are in clinical development including berzosertib (M6620, VX-970), which is currently being evaluated in combination with cytotoxic chemotherapies and radiation. Although clinical studies have examined plasma pharmacokinetics (PK) in humans, little is known regarding dose/exposure relationships and tissue distribution which may influence berzosertib’s therapeutic window. To understand these concepts, we extensively described the PK of berzosertib in mouse plasma and tissue. Methods: Dose proportionality was assessed using single IV doses of berzosertib (2, 6, 20 and 60 mg/kg) administered to female BALB/c mice. Mice were euthanized from 5 min to 24 h post-dose. Using a highly sensitive LC-MS/MS method, berzosertib was quantitated in red blood cells, plasma, and tissues. Non-compartmental analysis (NCA) was performed using Phoenix WinNonlin to determine relevant PK parameters. Dose linearity was assessed by statistical comparison of dose-normalized plasma Cmax and dose-normalized tissue AUC across the four dose levels. Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons test. Berzosertib plasma protein binding was assessed in vitro from 300 ng/mL to 100 μg/mL using rapid equilibrium dialysis. An extensive PK study was also conducted in tumor-bearing mice to assess distribution by quantitating drug in tissues. Results: The dose proportionality study indicated that both plasma Cmax and AUC increase with dose, and dose-normalized concentration vs time profiles suggested nonlinear increases in exposure based on different terminal slopes. At the two highest doses, dose-normalized AUC in each tissue were greater than the exposure generated at the two lowest doses. However, log transformations of AUC0-360 plotted vs dose generated a slope of 0.886 (95% CI: 0.718 - 1.054), suggesting a linear relationship. The fraction of unbound berzosertib in mouse plasma increased at concentrations above 10 μg/mL, which is a concentration exceeded at the two highest doses and where nonlinearity in exposure is observed. Tissue analysis revealed the following tissues had a tissue/plasma coefficient >1: bone marrow, kidney, liver, spleen, lung, tumor, lymph nodes, thymus, and heart. Conclusion: IV berzosertib displayed nonlinear PK in mice. Increased doses of berzosertib were associated with greater than proportional increases in plasma and tissue exposure most likely due to saturation of plasma protein binding. Our results will help to understand preclinical pharmacodynamic and toxicity data and subsequent PBPK modeling to inform optimal dosing and clinical deployment of berzosertib. Citation Format: Joshua J. Deppas, Brian F. Kiesel, Jianxia Guo, Robert A. Parise, Christopher J. Bakkenist, Jan H. Beumer. Non-linear IV pharmacokinetics of the ATR inhibitor berzosertib (M6620) in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7171.

First-in-Human Study of the Ataxia Telangiectasia and Rad3-Related (ATR) Inhibitor Tuvusertib (M1774) as Monotherapy in Patients with Solid Tumors.

Tuvusertib (M1774) is a potent, selective, orally administered ataxia telangiectasia and Rad3-related (ATR) protein kinase inhibitor. This first-in-human study (NCT04170153) evaluated safety, tolerability, maximum tolerated dose (MTD), recommended dose for expansion (RDE), pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of tuvusertib monotherapy. Ascending tuvusertib doses were evaluated in 55 patients with metastatic or locally advanced unresectable solid tumors. A safety monitoring committee determined dose escalation based on PK, PD, and safety data guided by a Bayesian 2-parameter logistic regression model. Molecular responses (MR) were assessed in circulating tumor DNA samples. Most common grade ≥3 treatment-emergent adverse events were anemia (36%), neutropenia, and lymphopenia (both 7%). Eleven patients experienced dose-limiting toxicities, most commonly grade 2 (n = 2) or 3 (n = 8) anemia. No persistent effects on blood immune cell populations were observed. The RDE was 180 mg tuvusertib QD (once daily), 2 weeks on/1 week off treatment, which was better tolerated than the MTD (180 mg QD continuously). Tuvusertib median time to peak plasma concentration ranged from 0.5 to 3.5 hours and mean elimination half-life from 1.2 to 5.6 hours. Exposure-related PD analysis suggested maximum target engagement at ≥130 mg tuvusertib QD. Tuvusertib induced frequent MRs in the predicted efficacious dose range; MRs were enriched in patients with radiological disease stabilization, and complete MRs were detected for mutations in ARID1A, ATRX, and DAXX. One patient with platinum- and PARP inhibitor-resistant BRCA wild-type ovarian cancer achieved an unconfirmed RECIST v1.1 partial response. Tuvusertib demonstrated manageable safety and exposure-related target engagement. Further clinical evaluation of tuvusertib is ongoing.

Abstract C162: Assessment of the potential for QTc prolongation by the investigational ATR inhibitor tuvusertib in patients with advanced solid tumors using integrated nonclinical and clinical assessments

Abstract Background: Tuvusertib (M1774) is a potent, selective, orally administered inhibitor of ataxia telangiectasia and Rad3-related protein kinase (ATR) with demonstrated antitumor activity in preclinical models. Tuvusertib is currently being evaluated in phase I and II trials as monotherapy and in combination with other DNA damage response inhibitors and immune checkpoint inhibitors. Assessment of an investigational agent’s potential to cause delayed ventricular repolarization (QTc prolongation) is essential in drug development. QTc prolongation is assessed using nonclinical studies and concentration-QTc (c-QTC) analysis of phase I clinical data across a wide dose range and/or a thorough QT/QTc (TQT) clinical study. Here, we assess tuvusertib’s potential to cause QTc prolongation by integrating nonclinical evaluations and clinical c-QTc analyses. Methods: Nonclinical evaluations included in vitro binding of tuvusertib to the ether-a-go-go-related gene (hERG) channel and telemetry of tuvusertib in male beagle dogs and minipigs. Pharmacokinetics-time-matched triplicate electrocardiograms were collected in patients with advanced solid tumors from the first-in-human (FIH) trial (NCT04170153) for tuvusertib 5−270 mg QD along with intermittent dosing regimens. Based on exploratory analyses, a linear mixed-effect model (Garnett C, et al. J Pharmacokinet Pharmacodyn 2018;45:383–97) was chosen to describe the relationship between tuvusertib plasma concentrations and selected heart rate (HR)-corrected QT using the Fridericia correction (QTcF). Results: In vitro hERG channel binding potency for tuvusertib 0.3–10.0 µM was calculated with a half-maximal inhibitory concentration (IC50) of 2.8 µM. Nonclinical telemetry studies showed no treatment effect on QT/QTc prolongation at doses of up to 3 mg/kg in male beagle dogs and minipigs. c-QTc analyses in 55 patients from the FIH trial revealed a small positive slope of drug effect (slope estimate [95% CI] = 0.00244 [0.000708, 0.00417] ms/(ng/mL)). This increase in model-predicted QTcF from baseline was not considered to be clinically meaningful (<20 ms threshold) at steady-state maximum concentration (Cmax) achieved with the recommended dose for expansion (RDE) of 180 mg QD 2w on/1w off (mean [upper bound of 95% CI] = 3.4 [5.5] ms) and up to 3 times the RDE (10.3 [16.5] ms). Concentration-HR modeling found no relationship between tuvusertib concentration and HR, suggesting the absence of an effect. Conclusions: Integrating nonclinical and clinical assessments revealed no clinically meaningful effect of tuvusertib on QTc at concentrations achieved with the RDE and up to 3-times higher concentrations. Citation Format: Jatinder Kaur Mukker, Timothy A. Yap, Anthony W. Tolcher, Johann S. de Bono, Ruth Plummer, Rohan Kulkarni, Han Witjes, Paul Matthias Diderichsen, Axel Krebs-Brown, Rainer Strotmann, Zoltan Szucs, Ioannis Gounaris, Karthik Venkatakrishnan. Assessment of the potential for QTc prolongation by the investigational ATR inhibitor tuvusertib in patients with advanced solid tumors using integrated nonclinical and clinical assessments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C162.

Investigation of ATR Inhibitor VX970 as a Radiosensitizer in Colorectal Cancer Cells

Colorectal cancer (CRC) is the second leading cause of combined cancer-related mortality in males and females in the U.S. Traditional treatment of locally advanced rectal cancer consists of neoadjuvant chemoradiation followed by surgery and adjuvant chemotherapy. Emerging data suggests that higher response rates can be achieved with total neoadjuvant therapy (TNT) where delivery of all chemotherapy and radiation therapy (RT) occurs prior to surgery. In addition, for patients with a complete response to TNT, non-operative management (NOM) can be considered. However, despite the use of TNT, pathologic complete response rates remain below 40% and NOM is only achieved in approximately 50% of rectal cancer patients. A strong need remains for more active anti-cancer therapies in rectal cancer to both reduce pelvic recurrence and facilitate NOM. Here, we tested the hypothesis that inhibition of the ataxia telangiectasia and Rad3-related protein kinase (ATR), a critical regulator of cellular DNA damage response, could increase the sensitivity of CRC to RT. VX970, a highly potent and selective ATR inhibitor, was investigated as a radiosensitizer in SW48 and LoVo CRC cell models. In vitro, IC50 of VX970 was assessed by alamarBlue cytotoxicity assay, while radiosensitivity was revealed by radiation clonogenic assays (0, 2, 4, 6, 8 Gy). ATR activity was determined by p-Chk1 using immunoblotting, and cell cycle distribution was analyzed by propidium iodide flow cytometry. CRC xenografts were generated using both LoVo and SW48 cells injected in the left flanks of athymic nude mice to explore the radiosensitizing effects of VX970 in vivo. Tumors were allowed to grow to 100-150 mm3, and the mice were randomized into multiple groups [vehicle alone, RT alone (10 Gy/5 fractions), VX970 alone, and VX970+RT]. Mouse weights and tumor size were measured three times weekly. Comparison of treatment groups was performed using the log-rank test with P<0.05 considered significant. The IC50 concentrations of VX970 on SW48 and LoVo cells were about 500 and 100 nM, respectively. VX970 at doses of 3 nM did not alter the viability of CRC cells, but significantly sensitized CRC cells to radiotherapy (P<0.05), with DER of 1.43 and 1.59, respectively, in SW48 and LoVo cells. VX970 efficiently attenuated p-Chk1 expression and significantly abrogated radiation induced G2/M cell cycle arrest (P<0.05). In addition, VX970 in combination with radiotherapy significantly prolonged tumor growth delay of CRC xenografts compared to radiation alone (P<0.05), with minimal toxicity observed. Inhibition of the ATR-Chk1 pathway by targeting ATR kinase with VX970 sensitizes CRC to radiotherapy in vitro and in vivo. Our findings support that ATR inhibition by VX970 is a promising new approach to improve the therapeutic ratio of radiotherapy for patients with CRC and warrants further clinical testing.

A phase 2 single-arm study of berzosertib in combination with irinotecan in patients with progressive TP53 mutant gastric and gastro-esophageal junction cancer.

4044 Background: TP53 mutations are reported in 30-70% of patients with metastatic/unresectable gastric (G) and gastro-esophageal junction (GEJ) adenocarcinoma and are associated with a poor prognosis. Novel therapeutics are needed. Gastrointestinal cancer cell lines with TP53 mutations demonstrate reliance on the ataxia telangiectasia and Rad3-related protein kinase (ATR) axis for DNA damage repair (DDR) in vitro. The ATR axis is triggered by single-strand DNA breaks and thus combining ATR inhibitors with topoisomerase I (TopI) inhibitors may have a synergistic effect, particularly in patients with TP53 mutant tumors. Irinotecan, a TopI inhibitor, is an established cytotoxic therapy for gastric and GEJ adenocarcinoma. We evaluated irinotecan in combination with the ATR inhibitor berzosertib (formerly M6620) in patients with TP53-mutated gastric and GEJ adenocarcinoma. Methods: This was a single-arm phase II study in third-line patients with G/GEJ cancers possessing TP53 mutations (exon 2 and exons 4-11). Patients received irinotecan 180 mg/m2 IV and berzosertib 270 mg/m2 IV on days 1 and 15 every 28 days. The primary endpoint was overall response rate (ORR) (Ha: 35% and Ho:15%). A subset of patients underwent biopsies on C1D1 post-irinotecan and C2D2 to evaluate the expression of DDR biomarkers Y-H2AX, KAP1 p-Ser 824, and p-ATR. Results: A total of 17 patients were enrolled. Of 16 efficacy-evaluable patients, one experienced a partial response. The ORR was 6.2% thus the primary endpoint was not met. The disease control rate was 56.2%. Median progression-free survival was 4.01 months [95% CI 2.07-Not Reached]. Median overall survival was 6.21 months [95% CI 4.83-8.61]. No new safety signals were observed with the experimental treatment combination and the most common ≥ grade 3/4 treatment-related adverse events were neutropenia (4/17, 23.5% patients), anemia (3/17, 17.6%), febrile neutropenia (2/17, 11.8%), and diarrhea (2/17, 11.8%). No grade 5 events occurred. Conclusions: Although the primary endpoint of ORR was not met, the combination of irinotecan and berzosertib in patients with TP53-mutated G/GEJ adenocarcinoma was well-tolerated and demonstrated anti-tumor activity comparable to other agents with published later-line data (e.g. ramucirumab+paclitaxel, TAS-102). Analyses of pharmacodynamic biomarkers of DNA damage repair are in process. While further clinical development of berzosertib is not planned, other ATR inhibitors are being explored in combination with irinotecan in gastrointestinal malignancies, including G/GEJ cancer. Clinical trial information: NCT03641313 . [Table: see text]

LATIFY: Phase 3 study of ceralasertib + durvalumab vs docetaxel in patients with locally advanced or metastatic non-small-cell lung cancer that progressed on or after anti-PD-(L)1 and platinum-based therapy.

TPS9161 Background: Following progression on first- or second-line immunotherapy ± platinum-based chemotherapy (CT), the prognosis for patients (pts) with metastatic non-small-cell lung cancer (NSCLC) is poor with limited alternative options to docetaxel. Ceralasertib is a selective inhibitor of Ataxia Telangiectasia and Rad3-related (ATR) protein kinase, which is activated in response to DNA damage. Clinical data suggest that ceralasertib may sensitize tumors to immunotherapy by biasing T cells to an immune-effective phenotype. Durvalumab is a selective, high-affinity human IgG1 monoclonal antibody that binds programmed cell death ligand-1 (PD-L1) and inhibits PD-L1-mediated suppression of T-cell activation. Combining ceralasertib with durvalumab may amplify the antitumor immune response, and potentially lead to durable tumor control. In the ongoing phase 2 HUDSON study in pts with locally advanced or metastatic NSCLC who progressed on anti-PD-(L)1 therapy and platinum-doublet regimen, ceralasertib + durvalumab showed promising efficacy with median progression-free survival (PFS) of 6.0 mos (80% CI, 4.6–7.5) and median overall survival (OS) of 15.9 mos (80% CI, 14.1–20.3). Methods: LATIFY is a phase 3, open-label, randomized, multicenter study in pts with NSCLC (NCT05450692). Key inclusion criteria are age ≥18 years; ECOG performance status 0–1; documented EGFR and ALK wild-type, and tumor cell PD-L1 status; and adequate organ and bone marrow function. Stable brain metastases are allowed. Pts should be eligible for second- or third-line therapy, and must have received an anti-PD-(L)1 therapy and a platinum-containing doublet regimen for locally advanced or metastatic NSCLC either separately or in combination, but no other prior therapies. Key exclusion criteria include mixed small-cell lung and NSCLC histology, unresolved toxicities of Grade ≥2 (NCI CTCAE v5.0) from prior therapy, active or prior autoimmune or inflammatory disorders, &gt; 1 line of prior anti-PD-(L)1 therapy (alone or in combination), and &gt; 1 line of platinum-based CT in a metastatic setting. Pts are randomized 1:1 to receive either oral ceralasertib 240 mg twice daily on Days (D) 1–7 with IV durvalumab 1500 mg on D8 (28-day cycle) or IV docetaxel 75 mg/m2 (D1, 21-day cycle). The primary objective is to assess efficacy by OS. Secondary objectives include evaluating efficacy by PFS, objective response rate and disease control rate by RECIST v1.1; duration of, and time to, response; time to second progression or death; OS at 12 mos; time to deterioration of health-related quality of life and physical function; determining the pharmacokinetics of ceralasertib; and assessing safety. Approximately 580 pts will be recruited from around 21 countries in the Americas, Europe, and Asia Pacific. Clinical trial information: NCT05450692 .

Selective inhibition of ATM-dependent double-strand break repair and checkpoint control synergistically enhances the efficacy of ATR inhibitors.

Ataxia Telangiectasia and Rad3-Related Protein (ATR) kinase regulates a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell cycle checkpoints controlled by Ataxia Telangiectasia Mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1 phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell cycle checkpoints and DSB repair, lowered the p53 protective barrier and extended the life of ATR inhibitor induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Further, a combination study in 26 patient-derived xenograft models of triple negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared to single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.

Abstract CT272: Pharmacodynamic and immunophenotyping analyses of ATR inhibitor M1774 in a Phase I study in patients with solid tumors (DDRiver Solid Tumors 301)

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase plays a critical role in the DNA damage response. M1774, a potent, selective, orally administered ATR inhibitor with antitumor activity in preclinical models, was evaluated in Part A1 of an open-label, single-arm study (NCT04170153) for safety, tolerability, maximum tolerated dose, pharmacokinetics (PK) and pharmacodynamics (PD). M1774 monotherapy in patients with advanced solid tumors was well-tolerated and the totality of evidence, including quantitative model-based analyses, suggested the recommended dose for expansion (RDE) as 180 mg QD 2 weeks on/1 week off.1 Here, we report findings of the M1774 PD and immunophenotyping analyses. Methods: M1774 PD was explored by assessing phosphorylation by ATR of CHK1 (p-CHK1) in tumor and of H2AX (γ-H2AX) in serial blood samples, stimulated ex vivo with the radiomimetic 4-Nitroquinoline N-oxide or Dimethyl sulfoxide as control. A flow cytometry quantitative assay was used to measure γ-H2AX in the CD45+ lymphocytes fraction. The effect of M1774 on the immunophenotype was explored by flow cytometry. Blood samples were collected at baseline, 3 and 24 hours after first M1774 administration on day 1 of cycle 1 for the γ-H2AX analysis, and on Days 1 and 15 of Cycles 1 and 2 before treatment for immunophenotyping. Results: Preclinical tumor tissue-blood bridging PD analyses in a mouse model demonstrated that the inhibition of γ-H2AX in lymphocytes highly correlated with inhibition of p-CHK1 in tumor. Clinical data of γ-H2AX levels and immunophenotyping were generated for the blood samples collected from the 55 participants of Part A1 of the study. Exploratory PK-PD analysis using γ-H2AX levels 3 h post-dose on day 1 across the doses predicted target inhibition &amp;gt;80% for doses ≥130 mg, suggesting target engagement. The levels of ɣ-H2AX at 24 h after first dose intake were variable and mean levels rebounded to baseline value. M1774 treatment did not cause any significant and consistent change in the levels of all explored immune cell subsets at the tested dose levels, including myeloid-derived suppressor cells, T and B lymphocytes, monocytes, and natural killer cells. Conclusions: PD analyses showed that M1774 efficiently inhibited ATR at the RDE without impacting the immunophenotype. 1TA Yap, et al. Ann Oncol. 2022; 33(suppl_7): S197-S224. Citation Format: Ruth Plummer, Anthony W. Tolcher, Timothy A. Yap, Giuseppe Sessa, Jatinder K. Mukker, Annick Seithel-Keuth, Christine Hicking, Zoltan Szucs, Ioannis Gounaris, Giuseppe Locatelli, Johann S. de Bono. Pharmacodynamic and immunophenotyping analyses of ATR inhibitor M1774 in a Phase I study in patients with solid tumors (DDRiver Solid Tumors 301) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT272.

ATR Inhibitors in Platinum-Resistant Ovarian Cancer

Platinum-resistant ovarian cancer (PROC) is one of the deadliest types of epithelial ovarian cancer, and it is associated with a poor prognosis as the median overall survival (OS) is less than 12 months. Targeted therapy is a popular emerging treatment method. Several targeted therapies, including those using bevacizumab and poly (ADP-ribose) polymerase inhibitor (PARPi), have been used to treat PROC. Ataxia telangiectasia and RAD3-Related Protein Kinase inhibitors (ATRi) have attracted attention as a promising class of targeted drugs that can regulate the cell cycle and influence homologous recombination (HR) repair. In recent years, many preclinical and clinical studies have demonstrated the efficacy of ATRis in PROC. This review focuses on the anticancer mechanism of ATRis and the progress of research on ATRis for PROC.

Arbidol inhibits human esophageal squamous cell carcinoma growth in vitro and in vivo through suppressing ataxia telangiectasia and Rad3-related protein kinase.

Human esophageal cancer has a global impact on human health due to its high incidence and mortality. Therefore, there is an urgent need to develop new drugs to treat or prevent the prominent pathological subtype of esophageal cancer, esophageal squamous cell carcinoma (ESCC). Based upon the screening of drugs approved by the Food and Drug Administration, we discovered that Arbidol could effectively inhibit the proliferation of human ESCC in vitro. Next, we conducted a series of cell-based assays and found that Arbidol treatment inhibited the proliferation and colony formation ability of ESCC cells and promoted G1-phase cell cycle arrest. Phosphoproteomics experiments, in vitro kinase assays and pull-down assays were subsequently performed in order to identify the underlying growth inhibitory mechanism. We verified that Arbidol is a potential ataxia telangiectasia and Rad3-related (ATR) inhibitor via binding to ATR kinase to reduce the phosphorylation and activation of minichromosome maintenance protein 2 at Ser108. Finally, we demonstrated Arbidol had the inhibitory effect of ESCC in vivo by a patient-derived xenograft model. All together, Arbidol inhibits the proliferation of ESCC in vitro and in vivo through the DNA replication pathway and is associated with the cell cycle.

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

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

494TiP Phase Ib study of elimusertib (ATRi; BAY 1895344) in combination with niraparib (PARPi) in patients with advanced solid tumors

An effective DNA damage response (DDR) is essential for cell survival. Elimusertib inhibits the ataxia-telangiectasia and RAD3-related (ATR) protein kinase, which is activated in response to DNA damage and mediates cell cycle arrest to maintain genomic integrity. Niraparib inhibits poly (ADP-ribose) polymerases 1/2 (PARP-1/2), which bind damaged regions of DNA and facilitate proper repair. Evidence suggests that combined inhibition of parallel DDR pathways act synergistically to improve anti-tumor responses, and the current study begins a clinical investigation of this hypothesis (NCT04267939).

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

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

1534P Phase II study of berzosertib + topotecan in patients with relapsed platinum (Pt)-resistant SCLC (DDRiver SCLC 250): Japanese safety run-in

Berzosertib is a potent, first-in-class selective inhibitor of ataxia telangiectasia and Rad3-related (ATR) protein kinase. Berzosertib is well tolerated and synergises with topotecan (TOP) in patients with advanced solid tumours.

Abstract 3971: Genome-wide CRISPR/Cas9 screening reveals ATR as a therapeutic target that overcomes osteosarcoma chemoresistance

Abstract Osteosarcoma (OS) is the most common bone cancer in pediatric patients. Therapeutic target identification in OS has been extremely challenging due to the high degree of heterogeneity in its genetic profile and lack of specific oncogenic driver genes. Despite extensive research efforts, the treatment and survival outcomes for OS patients have stagnated over the past 30 years. OS remains a lethal cancer due to metastasis and development of resistance to current chemotherapies. Study of chemo-resistance mechanisms and identification of novel targets that can be used in combination therapy are therefore critical to improve OS treatment. The combination of gene essentiality concept and clustered, regularly interspaced, short palindromic repeats-associated nuclease Cas9 (CRISPR/Cas9) functional knockout screen is an ideal new strategy to address the genetic complexity and drug-resistance in OS. A gene is considered essential if it is critical for reproductive success in an organism under specified condition. The recent introduction of genome editing using the CRISPR/Cas9 system with pooled library of guided RNAs (gRNAs) makes it feasible to systematically identify the genes required for the proliferation. By comparing the fold change of gRNA abundance in the surviving population of cells relative to the initial population, the essential genes under different settings can be identified.As a novel therapeutic approach for identifying combination therapies for OS, we conducted genome-wide (GW) CRISPR/Cas9 screen in SaOS2 OS cell lines parallel with RNA-sequencing in different conditions (control, Cisplatin, Doxorubicin), followed by gene essentiality score calculation. Cisplatin (CIS) and Doxorubicin (DOX) combination is among those commonly used as first-line therapies for OS treatment. The objective of our studies is to bring forth new perspectives in target identification and chemo-resistance study for OS. The gene targets and pathways that become more essential for OS cell proliferation in the presence of chemo drugs were revealed. Drugs specific for some of these target could then be added to the established CIS or DOX regimen to overcome existing drug resistance. On the other hand, the genes that become less essential after treatment could provide additional chemo-resistant mechanisms. Multiple gene targets were identified in the screen. Among these, we’ve successfully identified and validated Berzosertib, an ataxia telangiectasia and Rad3-related protein kinase inhibitor (ATRi), as a synergistic lethality partner to Cisplatin in vitro for OS treatment. Efficacy and safety measurement of Berzosertib and Cisplatin combination therapy in an OS xenograft mouse model and in several OS patient-derived xenograft models is in progress. Citation Format: Shan Tang, Karen E. Pollok, Pankita H. Pandya, Ryan D. Roberts, Xue Wu, Lang Li. Genome-wide CRISPR/Cas9 screening reveals ATR as a therapeutic target that overcomes osteosarcoma chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3971.