ATR Inhibitor Research Articles

Abstract 3369: Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage

Abstract Background: Endometrial cancer (EC) is the most common gynecologic malignancy with increasing incidence and mortality in the U.S. Among the four subtypes characterized by the Cancer Genome Atlas, the serous-like copy number high (CNH) subtype which occurs in 15-25% of EC is associated with poor prognosis and platinum resistance, requiring alternative treatment options. ~85% CNH EC cases have impaired G1/S checkpoint regulation, making cells rely on ATR-mediated G2/M cell cycle checkpoints for optimal DNA replication and repair. Additionally, over 80% of ECs exhibit PI3K pathway activation (e.g., PIK3CA mutations), linked to enhanced DNA repair and drug resistance. Therefore, we hypothesized that dual ATR and PI3Kα inhibition would enhance DNA damage, increasing cell death in EC. Methods: CNH EC cell lines included platinum-resistant (KLE, MFE280, HEC1A, ARK2) as well as platinum-sensitive (ARK1) lines used to evaluate the activity of ATR inhibitor (ATRi) camonsertib and PI3Kα inhibitor (PI3Kαi) inavolisib. Cell growth was assessed by 3-day XTT and 9-12-day colony-forming assays. The degree of combination synergy, additivity, or antagonism was calculated using SynergyFinder with a reference highest single agent (HSA) model. An HSA synergy score > 10 indicates synergy, -10 to 10 suggests additivity and < -10 represents antagonism. DNA damage endpoints were measured by alkaline comet assay and immunoblotting for γ-H2AX. Data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P < 0.05 was statistically significant. Results: All tested EC cell lines showed sensitivity to clinically attainable doses of ATRi monotherapy (IC50 0.09 - 0.98 μM), but varying sensitivity to PI3Kαi monotherapy (IC50 0.03 - 67.01 μM) unrelated to their PIK3CA mutation status. Combination treatment using clinically attainable concentrations (camonsertib < 6 μM; inavolisib < 10 μM) yielded additivity in all cell lines independent of platinum sensitivity (HSA synergy scores -0.8-10.0). Notably, increased cytotoxic effects were observed in both PIK3CA wild-type (KLE) and PIK3CA-mutant (MFE280 and HEC1A) platinum-resistant lines by colony-forming assays (4.8- to 15.4-fold decrease in colony-forming ability relative to ATRi; 1.5- to 3.5-fold decrease relative to PI3Kαi). ATRi and PI3Kαi combination induced greater DNA damage, evidenced by elevated mean comet tail moment relative to ATRi (increased 2.5- to 6.7-fold; P < 0.01) or PI3Kαi (increased 2.1- to 5.6-fold; P < 0.01) regardless of PIK3CA mutation status. Increased γ-H2AX levels were found in HEC1A cells with combination treatment (3.2-fold increase relative to ATRi; 16-fold increase relative to PI3Kαi). Conclusion: Our results suggest that dual inhibition of ATR and PI3K pathways induces greater cell death by increasing DNA damage in platinum-resistant EC cells independent of PIK3CA mutation status. Citation Format: Kristen R. Ibanez, Tzu-Ting Huang, Sotirios Sotiriou, Yvonne G. Lin, Jung-Min Lee. Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage [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 3369.

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.

Abstract 3900: A high-throughput platform identifies novel drug combinations towards acute myeloid leukemia therapy

Abstract Molecularly targeted therapies have reshaped oncology in recent decades, though not all patients are eligible for such treatments and resistance mechanisms limit clinical benefit. In response, combination therapies, which can induce potent synthetic lethalities, bypass drug resistance, and expand treatment options, are increasingly being developed. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with broadly testing two or more agents. Thus, there is a practical need for methodologies that prioritize test compounds and technologies for rapid and efficient screening. Towards these ends, we have developed a novel combination drug screening workflow that integrates machine learning-based drug synergy predictions with a high throughput droplet microfluidics-based screening platform. Our physical system, or FlowMatrix, consists of 9,216 nano wells arrayed in a 96 × 96 grid in which cells are incubated and drug compounds delivered via emulsified droplets along both its rows and columns to facilitate combination screening. Following multi-day incubation, live cells are imaged with fluorescence microscopy and drug combination synergies are calculated from cellular viabilities. In our current setup, which includes four dose drug treatments with replicates and controls, we capture complete data (control, single agent, and 4 × 4 drug-drug combination viability measurements) for up to 100 unique drug combinations within the 9,216 dual droplet loaded wells of one FlowMatrix. Our to-date screening efforts have focused on acute myeloid leukemia (AML) specifically, where we have profiled 12,700 AML cell line-drug combinations in our system. This encompasses >3,700 unique drug combinations profiled in 7 distinct AML cell lines with 183 total matrix runs. Among the strongest hits observed in our screens and confirmed with validation studies (~65% confirmation rate) are combinations currently being tested in clinical trials for other cancers and established therapies paired with non-cancer indicated drugs. In particular, we are interrogating combinations involving CDK12 inhibitors, which affect the transcription of DNA repair proteins, splicing, and cancer cell progression, including unexpected partner drugs that potentiate the effects of CDK12 blockade in AML cell lines. We have uncovered additional unexpected synergistic combinations with KIT inhibitors in RUNX1-RUNX1T1 altered AML lines, ATR inhibitors (including an established combination with Gemcitabine), and the BCL-2 inhibitor Venetoclax, including known partnerships with chemotherapies (e.g. Decitabine and Daunorubicin) and other targeted inhibitors. Deeper studies are underway to further validate and characterize these candidates towards expanding treatment options for AML. Ultimately, our platform may radically enhance drug combination screening potential. Citation Format: Anthony R. Soltis, Boryana Zhelyazkova, Pascal Drane, Efstathios Eleftheriadis, Andrew Ventresco, David A. Weitz, Arlinda Lee, Anthony J. Iafrate. A high-throughput platform identifies novel drug combinations towards acute myeloid leukemia therapy [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 3900.

Abstract 606: The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with rapid disease progression and poor patient survival. Therapeutic options for SCLC remain a challenge due to limited understanding of both the biology of the disease and the major determinants of treatment response. Key transcription factors associated with unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Previously, we demonstrated that onvansertib, a highly specific inhibitor of the PLK1 Ser/Thr kinase, was effective in SCLC preclinical models, leading to its ongoing clinical development for relapsed/refractory SCLC patients (NCT05450965). Here we further explored potential strategies to enhance onvansertib activity by assessing its in vitro and in vivo activity in combination treatments. Methods: Human SCLC cell lines were treated for 72 hr with onvansertib in combination with paclitaxel, lurbinectedin or the ATR inhibitors BAY1895344 and AZD6738. Effect on cell proliferation was analyzed by WST1 and CellTiterGlo assays. In vivo activity of onvansertib in combination with paclitaxel was tested in 3 SCLC patient-derived xenograft (PDX) models either cisplatin-sensitive (TKO5- ASCL1 subtype) or cisplatin-resistant (TKO2-ASCL1 subtype, TKO8-NEUROD1 subtype). Tumor-bearing nude mice were treated with vehicle, onvansertib (50 mg/kg, oral, 4 times/week), paclitaxel (15 mg/kg, IP, once a week) or the combination for 5 weeks. Mice were followed for tumor growth and survival (defined as time to reach tumor volume greater than 2000 mm3). Results: The combination of onvansertib and paclitaxel inhibited cell proliferation synergistically in cell lines from 3 SCLC subtypes (ASCL1, NEUROD1, and YAP1), while the combinations with lurbinectedin or ATR inhibitors were not synergistic. We further examined the combination of onvansertib and paclitaxel in SCLC PDXs. The combination exhibited potent anti-tumor activity, resulting in significantly greater tumor growth inhibition and increase in survival compared to the monotherapies in all 3 PDXs. The combination was well tolerated with no observed body weight loss. While onvansertib and paclitaxel had negligible single agent activity in cisplatin-resistant TK02 PDX and TKO8 PDX models, the combination induced durable complete responses in 25% and 100% of treated mice respectively. Conclusion: The PLK1 inhibitor onvansertib in combination with paclitaxel showed synergy in multiple SCLC cell lines in vitro and potent anti-tumor activity in cisplatin-sensitive and -resistant SCLC PDX models in vivo. Tumor tissue analysis is underway to detail the mechanisms of this interaction. Citation Format: John C. Schmitz, Guojing Zhang, Abbe Pannucci, Tod Smeal, Chu-Chiao Wu, Maya Ridinger, Taofeek K. Owonikoko. The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer [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 606.

Abstract 380: Co-downregulation of GRP78 and ATR enhances apoptosis in pancreatic ductal adenocarcinoma

Abstract Background: Targeting endoplasmic reticulum (ER) stress is a potential therapeutic strategy in preclinical models of pancreatic duct adenocarcinoma (PDAC). GRP78 is known as the master regulator of the unfolded protein response (UPR) pathway, regulating ER stress by activating the UPR pathway to avoid cell apoptosis and regulates the activation of UPR pathway signals by binding PERK. Overexpression of GRP78 in PDAC results in the retention of misfolded and unassembled proteins, as well as the accumulation of ROS, leading to the promotion of genomic instability. However, there has been limited research on targeting GRP78 as a therapeutic strategy in PDAC. In this study, we aimed to investigate the anticancer effects of BOLD-100, an inhibitor of GRP78, in PDAC. Methods: We used 9 PDAC cell lines (Capan-1, AsPC-1, HPAFII, MIA-PaCa2, Panc-1, Capan-2, SNU-213, SNU-324, SNU-2918). The mouse xenograft model of Capan-1 was established for in vivo study. BOLD-100 (GRP78 inhibitor), AZD6738 (ATR inhibitor), NAC (N-Acetylcysteine; ROS scavenger) and TUDCA (Tauroursodeoxycholic acid; ER stress inhibitor) were used. MTT assay, CFA assay, cell cycle analysis, RT-PCR, western blot, immunoprecipitation, DCF-DA staining and Annexin V assay were used to elucidate the action of BOLD-100. The combination of BOLD-100 with AZD6738 has been evaluated in both in vitro and in vivo model. Results: BOLD-100 suppressed the proliferation of PDAC cells and decreased the mRNA level of GRP78, which in turn disrupted the interaction between GRP78 and PERK. BOLD-100 increased ER stress and ROS, leading to activation of PERK, eIF2a, and CHOP. Activation of UPR pathway induced CHOP-dependent apoptosis and inhibited PDAC cell growth. Moreover, TUDCA reversed the ROS accumulation induced by BOLD-100, confirming that ER stress regulates ROS levels. The accumulation of ROS upregulated the active forms of ATR and CHK1, suggesting the activation of the DNA damage repair pathway. Notably, the treatment of NAC abrogated the activation of ATR-CHK1 axis by BOLD-100-induced ROS accumulation. AZD6738 synergized with BOLD-100 in in vitro and in vivo, by suppressing BOLD-100-induced ATR phosphorylation. Conclusion: GRP78 could serve as one of the potential therapeutic targets in PDAC. The combination of BOLD-100 and AZD6738 demonstrates a synergistic effect suggesting GRP78/ATR dual targeting as a promising therapeutic option for patients with PDAC. Citation Format: Su In Lee, Ah-Rong Nam, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Yoojin Jeong, Sea Young Choo, Hyo Jung Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. Co-downregulation of GRP78 and ATR enhances apoptosis in pancreatic ductal adenocarcinoma [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 380.

Abstract 591: Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1)

Abstract The genome DNA is consistently threatened by a variety of intrinsic and exogenous DNA damage factors. Cell cycle checkpoint is one important mechanism in response to DNA damage and maintaining genome stability. In tumor cells, gene alterations, such as CCNE1 amplification (encoding for Cyclin E1), result in G1/S checkpoint loss and replication stress, leading to high dependency on the G2/M checkpoint. PKMYT1 inhibits crucial checkpoint protein CDK1 activity by phosphorylating CDK1 at the residues of Thr-14 and Tyr-15 in the G2/M phase. In CCNE1 amplification cells, the inhibition of PKMYT1 leads to the reduction of pCDK1 and loss of G2/M checkpoint, consequently results in premature mitosis and the accumulation of DNA damage, then finally induces cell death. Here we identify a novel small molecular ZM-2322, which displays robust intracellular binding to PKMYT1 and inhibits the specific downstream phosphorylation of CDK1 Thr-14 with IC50 values of 14 nM and 29 nM, respectively. ZM-2322 strongly inhibits the proliferation of the HCC1569 cell line with CCNE1 high-level amplification and shows a >400 × selectivity folds over a CCNE1 wildtype cell line SKOV3. ZM-2322 also shows significant inhibition of tumor growth in a dose-dependent manner in the HCC1569 xenograft model, and the efficacy correlates well with PK and target inhibition. There is evidence showing that loss-of-function mutations of FBXW7, a cyclin E E3 ligase, lead to cyclin E accumulation and are synthetic-lethal with PKMYT1 inhibition. Consistently, a potent anti-proliferation of ZM-2322 is observed in DLD-1 FBXW7 -/- cells, but not DLD-1 parental cells. These data suggest a high potency of ZM-2322 on PKMYT1 inhibition and a potential good kinase selectivity which is further confirmed by using a kinase panel biochemical assay. As expected, when compared head-to-head with another clinical PKMYT1 inhibitor RP-6306 which shows inhibitory effect on 27 of 217 human kinases with inhibition rates >90% at 1 μM, ZM-2322 is found to be a much more PKMYT1-selective inhibitor and hits only several individual kinases under the same assay condition, suggesting a potentiality in better safety profile and higher dose tolerance. Furthermore, our data illustrates that in combination with gemcitabine, ZM-2322 elicits strong synergetic anti-proliferation activities on HCC1569 cells in vitro and results in tumor regression with a minor effect on body weight in vivo. Besides, a strong synergy effect is also observed in the combo of ZM-2322 and ATR inhibitor in HCC1569 in vitro. These data indicate that ZM-2322 has the potential of application expansion and good tolerance under continuous administration. Taken together, our data suggest that ZM-2322 is a potent and highly selective PKMYT1 inhibitor. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Baoying Cheng, Zhentao Li, Dongxing Zhu, Jianbin Xue, Liting Xue, Renhong Tang. Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1) [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 591.

Abstract 4539: ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics

Abstract Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway, and its dysfunction leads to cell genome instability and can result in tumorigenesis. In the situation of HR deficiency, DNA polymerase theta (Polθ) mediated microhomology-mediated end joining (MMEJ) is up-regulated to serve as a backup pathway for DSB repair. Several studies have proved that the inhibition of Polθ causes synthetic lethality with HR deficiency, and Polθ emerges as a potential DNA damage repair (DDR) target for the treatment of HR deficient tumors. In addition, there is evidence showing that the depletion of Polθ improves the sensitivity of some other DDR-related targeted therapies, chemotherapy, and radiotherapy. Here we identify a novel small molecular Polθ inhibitor, ZM-2311, which inhibits Polθ activity with an IC50 of 24 nM, and strongly inhibits cellular MMEJ pathway with a single nanomolar IC50. ZM-2311 illustrates a robust potency against tumor growth in a MDA-MB-436 SHLD2-/- xenograft model, suggesting potential monotherapy application in BRCA1 mutation patients with TP53BP1/Shieldin complex deficiency. Besides monotherapy, ZM-2311 elicits strong synergetic anti-proliferation activities in combination with PARP inhibitors on BRCA2-/- DLD-1 and endogenous HR deficient tumor cells in vitro and in vivo. Tumor regression is observed even at a low dosage, indicating a high potency of ZM-2311. As reversion mutation of HR genes such as BRCA1/2 mediated by the MMEJ pathway is reported to be a mechanism of PARP inhibitor resistance, ZM-2311 is evaluated and demonstrates its potential to overcome the resistance to PARP inhibitors. Furthermore, the combination of ZM-2311 with other DDR agents, such as ATR inhibitors, induces fatal DNA damage to HR deficient cells. Considering a low risk of hematotoxicity of Polθ inhibition, ZM-2311 may provide a new combination option for DDR agents. Besides, ZM-2311 was assessed in combination with different therapies respectively which functionally induce DNA damage, such as radiotherapy and chemotherapy. ZM-2311 renders tumor cells more sensitive to radiotherapy in both HR deficient cells and proficient cells in vitro, accompanying with the upregulation of Polθ expression after radiotherapy and an enhanced phosphorylation level of H2AX in the combination treatment. Similarly, ZM-2311 also achieves a robust synergetic effect in combination with chemotherapy in cell lines with different HR statuses. These results indicate a potential application expansion of ZM-2311 to HR-proficient tumors, which is expected to bring better efficacy and reduced hematotoxicity. Taken together, ZM-2311 has proved to be a highly potent Polθ inhibitor and has promising combination potential with multiple therapy strategies. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Shuo Qian, Liting Xue, Mengying Li, Zhengtao Li, Zhen Li, Jian Li, Renhong Tang. ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics [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 4539.

Abstract 7129: DNA polymerase beta expression in head & neck cancer modulates the poly-ADP-ribose-mediated replication checkpoint

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) impose a significant health burden, necessitating innovative therapeutic strategies to enhance treatment efficacy. Current standard treatments, including surgery, radiation therapy, and chemotherapy, often exhibit limited effectiveness and cause substantial side effects. This underscores the critical need for novel therapies that selectively target cancer cells, sparing normal tissue. Survival from HNSCC is poor, and importantly, a racial disparity between African Americans (AfAm) and European Americans (EuAm) has been known for some time. Ancestry Informative Markers (AIMs) are associated with altered mRNA expression of Polβ where higher expression of Polβ was observed among AfAm patients in comparison to EuAm patients. Our research focuses on DNA polymerase beta (Polβ) and its role in replication stress and response to chemotherapy and DNA damage response modifiers. Elevated expression of Polβ is linked to increased DNA damage and genomic instability, contributing to treatment resistance. In this regard, our investigations reveal a regulatory role for base excision repair (BER) proteins, including Polβ, in the cellular response to inhibitors of poly(ADP-ribose) glycohydrolase (PARG), an enzyme involved in poly(ADP-ribose) (PAR) degradation. Inhibition of PARG triggers a robust intra-S phase response, activating ATR and CHK1 signaling. Exploiting the synthetic lethality between PARG inhibition and ATR/CHK1 inhibition, we have demonstrated the ability to overcome Polβ overexpression mediated treatment resistance in HNSCC cells. Specifically, our in vitro studies demonstrate that the response to a combination of an ATR inhibitor and PARG inhibitor is impacted by Polβ expression in HNSCC cells, with synergistic effects surpassing the efficacy of the individual inhibitors. Similarly, the combination of a CHK1 inhibitor and PARG inhibitor proves effective in cells with elevated Polβ expression, offering a potential dual therapeutic strategy. These combinations exhibit promising potential for HNSCC treatment when Polβ expression is elevated. In conclusion, our findings propose a novel therapeutic paradigm for HNSCC, employing combination therapy with ATR or CHK1 inhibitors and PARG inhibitors. This approach offers a targeted strategy, presenting a promising avenue for the development of more effective and less toxic treatment modalities in the management of HNSCC and may help overcome resistance from elevated expression of Polβ. Citation Format: Md Maruf Khan, Anusha Angajala, Denise Y. Gibbs, Jeffrey C. Liu, Camille Ragin, Robert W. Sobol. DNA polymerase beta expression in head & neck cancer modulates the poly-ADP-ribose-mediated replication checkpoint [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 7129.

Abstract 7131: Base excision repair regulation of replication stress induced poly(ADP-ribose) checkpoints

Abstract PARP-inhibitors (PARPi) have been developed to treat homology-directed repair deficient cancers such as BRCA1/2-defective breast and ovarian tumors. However, acquired resistance to PARPi is now common, suggesting the need to identify new protein targets. To that end, a missed opportunity is found in the PARP1/PARP2 activation mechanism in response to DNA damage and replication stress and the resulting protein complexes and enzymes that depend on and coordinate PARP-mediated DNA repair processes. We have found that PARP1/PARP2 activation, whether from acute DNA damage, from replication stress or both triggers a temporal and coordinated interplay of base excision repair and single-strand break repair (BER/SSBR) proteins and enzymes. In turn, the recruitment of these BER and SSBR proteins suppresses PARP1/PARP2 activation, thereby functioning as a network of PARP1/PARP2 regulators, modulating the PARP-mediated response to replication stress. To that end, we find that loss of expression of these key BER/SSBR proteins releases a break on replication-stress induced PARP1/PARP2 activation, triggering un-restrained PARP1/PARP2 activation and elevated levels of replication-dependent poly(ADP-ribose) (PAR). We next confirmed that the loss of these downstream BER enzymes render cancer cells responsive to PARPi’s when the BRCA1/2 pathways are restored. Further, we show that BER expression regulates the cellular response to inhibitors of the PAR degrading enzyme PARG, suggesting that XRCC1-dependent BER proteins regulate replication-stress dependent PAR accumulation. The increase in PAR due to PARG inhibition, that is further regulated by BER, drives a strong intra-S phase response, indicated by activation of ATR and CHK1 signaling. As the checkpoint governed by ATR and CHK1 functions as a protective mechanism, we show that PARG inhibition is synthetically lethal with ATR and CHK1 inhibition and this may be explained by our demonstration of PARGi-mediated trapping of essential BER proteins at the site of damage and PAR accumulation. Together, these studies reveal a significant role for BER/SSBR proteins in regulating replication stress induced PARylation and the resulting intra-S phase checkpoint. Citation Format: Md Ibrahim, Md Maruf Khan, Rasha Q. Al-Rahaleh, Jennifer Clark, Faisal Hayat, Qingming Fang, Christopher A. Koczor, Marie E. Migaud, Robert W. Sobol. Base excision repair regulation of replication stress induced poly(ADP-ribose) checkpoints [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 7131.

Pharmacological degradation of ATR induces antiproliferative DNA replication stress in leukemic cells.

Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.

Discovery of Selective and Potent ATR Degrader for Exploration its Kinase‐Independent Functions in Acute Myeloid Leukemia Cells

AbstractATR has emerged as a promising target for anti‐cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase‐independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis‐targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53‐mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti‐proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti‐AML activity is regulated by the kinase‐independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.

Discovery of Selective and Potent ATR Degrader for Exploration its Kinase-Independent Functions in Acute Myeloid Leukemia Cells.

ATR has emerged as a promising target for anti-cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase-independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis-targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53-mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti-proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti-AML activity is regulated by the kinase-independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.

AKTing on R Loops Makes for an ATRactive Target in Ovarian Cancer Therapy.

High-grade serous ovarian carcinoma (HGSOC) is the deadliest subtype of ovarian cancer. While PARP inhibitors (PARPi) have transformed the care of advanced HGSOC, PARPi resistance poses a major limitation to their clinical utility. DNA damage checkpoint signaling via ATR kinase can counteract PARPi-induced replication stress, making ATR an attractive therapeutic target in PARPi-resistant tumors. However, ATR inhibitor (ATRi) efficacy in the clinic is low, emphasizing the need for suitable combination treatments. In this issue of Cancer Research, Huang and colleagues uncovered cytotoxic synergism between inhibition of the PI3K/AKT pathway and ATR based on high-throughput screening for ATRi drug combinations in PARPi-resistant HGSOC cells. Dual inhibition of ATR and AKT resulted in aberrant replication stress and cell death, which was attributed in part to impaired resolution of replication-stalling RNA:DNA hybrids (R loops). The authors identified the DNA/RNA helicase DHX9 as a clinically relevant candidate effector of R loop resolution in HGSOC. AKT interacted with and recruited DHX9 to R loops, where it complemented ATR in facilitating their removal. Underlining the therapeutic potential relevance of these findings, combined inhibition of ATR and AKT caused near complete tumor regression in HGSOC xenograft models, and elevated AKT/DHX9 levels correlated with poor survival in patients with HGSOC. Of note, the genotoxic consequences of dual ATRi/AKTi treatment extended beyond PARPi-resistant tumors and are likely to affect genome integrity beyond R loops. The work by Huang and colleagues thus provides compelling rationale for the exploration of combined targeting of the AKT and ATR pathways as a potentially broadly applicable treatment of advanced HGSOC. See related article by Huang et al., p. 887.

Triple-Negative Breast Cancer and Emerging Therapeutic Strategies: ATR and CHK1/2 as Promising Targets.

Worldwide, breast cancer is the most frequently diagnosed malignancy in women, with triple-negative breast cancer (TNBC) being the most aggressive molecular subtype. Due to the dearth of effective therapeutic options for TNBC, novel agents targeting key mechanisms and pathways in cancer cells are continuously explored; these include ATR inhibitors, which target the ATR kinase involved in the DNA damage response (DDR) pathway, and CHK1/2 inhibitors, which target the Checkpoint Kinase 1/2 (CHK1/2) involved in cell cycle arrest and DNA repair. ATR and CHK1/2 inhibitors show potential as prospective treatments for TNBC by focusing on the DDR and interfering with cell cycle regulation in cancer cells. Preliminary preclinical and clinical findings suggest that when combined with chemotherapy, ATR and CHK1/2 inhibitors demonstrate significant anti-proliferative efficacy against TNBC. In this article, we introduce ATR and CHK1/2 inhibitors as promising therapeutic approaches for the management of TNBC. Preclinical and clinical studies performed evaluating ATR and CHK1/2 inhibitors for the treatment of TNBC and associated challenges encountered in this context to date are reviewed.

Functionally-instructed modifiers of response to ATR inhibition in experimental glioma

BackgroundThe DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma.MethodsWe used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo.ResultsATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo.ConclusionIn conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.

The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

ATR signaling controls the bystander responses of human chondrosarcoma cells by promoting RAD51-dependent DNA repair

Purpose Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines. Methods Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage. Results Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells. Conclusion Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Abstract A034: Combination of ATR inhibitor (ATRi) and AKT inhibitor (AKTi) reduces tumor growth and prolongs survival in BRCA2 mutant (BRCA2m) PARP inhibitor (PARPi)-resistant high-grade serous ovarian cancer (HGSC)

Abstract A034: Combination of ATR inhibitor (ATRi) and AKT inhibitor (AKTi) reduces tumor growth and prolongs survival in <i>BRCA2</i> mutant (BRCA2m) PARP inhibitor (PARPi)-resistant high-grade serous ovarian cancer (HGSC)

Abstract B027: Repurposing ion channel drugs: the Na+ channel modifying drug DPI-201-106 significantly enhances the anti-tumor activity of multiple classes of drugs targeting DNA damage repair

Abstract B027: Repurposing ion channel drugs: the Na+ channel modifying drug DPI-201-106 significantly enhances the anti-tumor activity of multiple classes of drugs targeting DNA damage repair