High-throughput combination screening of Pidnarulex and other G-quadruplex ligands in multi-cell type tumor spheroids.

Background: The serine/threonine kinase STK11/LKB1 is the second most commonly altered tumor suppressor in NSCLC. Non-functional mutations or loss of LKB1 expression occur more frequently in NSCLC than other alterations; however, there are currently no effective treatment strategies for this subset of tumors. KRAS-mutant LKB1 deficient NSCLC tumors often also have alterations in KEAP1 or NRF2 gene, which activate the KEAP1/NRF2 pathway known to be involved in antioxidant response. Inhibitors of ATM and ATR, two key proteins in the DNA damage response (DDR) pathway, are currently undergoing clinical testing but there are no validated biomarkers established for identifying which subgroups of patients are more likely to benefit from treatment. Here we have identified that alterations of LKB1, and the KEAP1/NRF2 pathway, are associated with enhanced response to ATM and ATR inhibitors (ATMi and ATRi) as well as other inhibitors of the DDR and may be useful biomarkers for predicting therapeutic response. Methods: To investigate the impact of LKB1 loss and KEAP1/NRF2 pathway activation on DDR and replication stress, we first tested replication fork protection in LKB1 deficient cells (KL). DNA fiber assay showed a defect in fork protection in KL cells compared with LKB1 wild type cells (K). Also, RPPA analysis revealed an activation of ATR/Chk1/Cdk1/CyclinB1 axis as well as Wee1 activation in cells harboring LKB1 and/or KEAP1 loss (KL/KLK/KK). Therefore, to evaluate response to DDR inhibitors (DDRi) we analyzed the in vitro activity of ATM, ATR, Wee1 and PARP inhibitors in NSCLC murine cell lines with or without knock out of LKB1 and/or KEAP1. In these cells, the loss of LKB1 and/or KEAP1 significantly sensitized to AZD0156 (ATMi), AZD6738 (ATRi) and AZD1775 (Wee1i) relative to cells with intact LKB1 and KEAP1. Next, we investigated whether the activity of ATR and ATMi in KL, KK or KLK tumor cells could be enhanced by the addition of a PARP inhibitor (olaparib). Although all NSCLC cells were resistant to the PARP inhibitor olaparib when used as a single agent, treatment of LKB1, KEAP1 or LKB1 plus KEAP1 deficient cells with the combination of olaparib plus ATM or ATR inhibitors significantly enhanced the antitumor cell activity of ATM or ATR inhibitors alone in vitro. We confirmed these data in an additional panel of LKB1 deficient NSCLC human cell lines treated with a broad spectrum of ATR and ATM inhibitors. In all human cell lines re-expression of LKB1 clearly reduced the sensitivity to ATR inhibition. LKB1 loss was also associated with sensitivity to PARP and ATM inhibitors, although these effects seemed to be less significant compared with ATR inhibitors. Interestingly, in vivo experiments performed in K, KL and KLK syngeneic models as well as PDX models showed greater response to ATRi and Wee1i monotherapy only in KLK but not in K or KL tumor models. Conclussions: Tumors with LKB1 deficiency or KEAP/NRF2 mutations are typically resistant to standard chemotherapy drugs and immunotherapy. Our data indicate that LKB1 and KEAP1/NRF2 loss significantly enhance the sensitivity to ATR, ATM and Wee1 inhibitors in vitro while only ATR and Wee1 inhibitor show significant tumor growth impairment in syngeneic and PDX KLK models. Thus, we have identified that NSCLC tumors bearing STK11 and KEAP1/NRF2 mutations are highly sensitive to DDR inhibitors and that genes may serve as biomarkers for selecting appropriate patients for treatment alone or in combination, such as PARPi, chemo or immunotherapy. Citation Format: Ana Galan-Cobo, Marlese Pisegna, Kavya Ramkumar, Alissa Poteete, Sungnam Cho, Fahao Zhang, You-Hong Fan, Qi Wang, Lixia Diao, Katharina Schlacher, Jing Wang, Lauren A Byers, John V. Heymcach. LKB1 deficiency and KEAP1/NRF2 pathway alterations as biomarkers of response for ATR and ATM inhibitors and other inhibitors of DNA damage response (DDR) in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A096. doi:10.1158/1535-7163.TARG-19-A096

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that ATM and PARP inhibitors showed remarkable synergy, which depends on the dominant negative function of ATM inhibition. Moreover, to provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting-based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage-signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Additionally, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. More recently, we have adopted dTAG technology for the investigation of many essential DDR and replication proteins, which provide mechanistic insights into the roles of these essential proteins in genome maintenance. Additionally, we have expanded FACS-based screens for the studies of other cancer-associated pathways and explored key biological processes critical for cancer progression in vivo. These studies will facilitate the development of better therapies for cancer patients. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1481.

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that ATM and PARP inhibitors showed remarkable synergy, which depends on the dominant negative function of ATM inhibition. Moreover, to provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting–based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage–signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Additionally, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. More recently, we have adopted dTAG technology for the investigation of many essential DDR and replication proteins, which provide mechanistic insights into the roles of these essential proteins in genome maintenance. Additionally, we have expanded FACS-based screens for the studies of other cancer-associated pathways and explored key biological processes critical for cancer progression in vivo. These studies will facilitate the development of better therapies for cancer patients. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B008.

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that ATM and PARP inhibitors showed remarkable synergy, which depends on the dominant negative function of ATM inhibition. Moreover, to provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting–based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage–signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Additionally, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. More recently, we have adopted dTAG technology for the investigation of many essential DDR and replication proteins, which provide mechanistic insights into the roles of these essential proteins in genome maintenance. Additionally, we have expanded FACS-based screens for the studies of other cancer-associated pathways and explored key biological processes critical for cancer progression in vivo. These studies will facilitate the development of better therapies for cancer patients. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr A009.

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results provide a better understanding of DDR pathways, which may facilitate the use of these DDR-targeting agents in cancer therapy. Moreover, To provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting-based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage-signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6189.

DDR kinase inhibition causes hypersensitivity to Taxol through caspase-3 activation.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Parallel In Vivo and In Vitro Melanoma RNAi Dropout Screens Reveal Synthetic Lethality between Hypoxia and DNA Damage Response Inhibition

Defective DNA damage repair, leading to genomic instability, is a common event during tumorigenesis. Despite enabling the persistence of mutations, any of which can confer a growth advantage to the nascent tumor, these defects place an Achilles Heel reliance on remaining repair pathways for survival from DNA damage. The protein kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) are key mediators of a DNA damage response activated by DNA damage during the S and G2 phases of cell cycle. Together they signal a series of cellular responses including activation of checkpoints and repair by homologous recombination. Loss of ATM pathway function frequently occurs in cancer, commonly from loss of function mutations in the tumor suppressor, p53, a substrate of ATM. This leads to a reliance on ATR that can be exploited for therapeutic benefit. Activation of ATR, by generation of S-phase DNA damage (replication stress, [RS]), can arise from treatment with DNA damaging drugs and certain targeted therapies such as inhibitors of poly ADP ribose polymerase (PARP). PARP is an enzyme involved in several DNA repair pathways, including base excision repair. Some PARP inhibitors have been shown to form an irreversible complex with DNA, potentially generating a direct RS lesion. While initial data indicates that inhibition of ATR and PARP is synergistic in some cancer cells, a comprehensive assessment has not been reported. Inhibition of ATR was cytotoxic in combination with PARP inhibitors against many cancer, but not non-cancer, cells. This effect was observed with multiple PARP inhibitors irrespective of their potential to form a DNA complex. In large cell panels of over 100 cancer cell lines, greater synergy was observed for the combination of an ATR and PARP inhibitor in cell lines with mutation of the TP53 gene. This was confirmed in isogenic cell lines depleted for ATM or p53, and is consistent with the profile of ATR and DNA damaging drug combinations. Furthermore, a triple combination of a PARP inhibitor, ATR inhibitor and the DNA damaging drug, cisplatin, retained cancer cell specific cytotoxic activity. In vitro dose-scheduling studies with the doublet of a PARP and ATR inhibitor showed optimal activity was achieved with transient concurrent exposure to both agents. This schedule contrasts with that for ATR inhibitors in combination with DNA damaging drugs where sequential dosing was optimal. In a mouse xenograft model concurrent dosing on a twice-weekly schedule was effective and well-tolerated. These data demonstrate the potential of combining ATR and PARP inhibitors in patients with p53 defective tumors. An optimal dose schedule was defined from cell and mouse studies. Together the data support clinical evaluation of ATR and PARP inhibitor combinations. Citation Format: John Pollard, Phil Reaper, Adele Peek, Stuart Hughes, Hakim Djeha, Steven Cummings, Karen Larbi, Marina Penney, Jim Sullivan, Darin Takemoto, Chris DeFranco. Pre-clinical combinations of ATR and PARP inhibitors: Defining target patient populations and dose schedule. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3711.

Simple SummaryDefective DNA damage response (DDR) is a hallmark of cancer leading to genomic instability. Up to 15–20% of colorectal cancers carry alterations in DDR. However, the role of DDR alterations as a prognostic factor and as a therapeutic target must be elucidated. To date, disappointing results have been obtained in different clinical trials mainly due to poor molecular selection of patients. Several challenges must be overcome before these compounds may have an impact on colorectal cancer. For instance, although some preclinical evidence showed the vulnerability of a subset of CRCs to PARP inhibitors, no specific clinical or molecular biomarkers have been validated to select patients. Moreover, different DDR alterations may not equally confer platinum sensitivity in CRC patients. Further efforts are needed in both preclinical and clinical settings to exploit DDR alterations as therapeutic targets and to eventually discover PARP or other DDR inhibitors (e.g., Wee1) with clinical benefit on colorectal cancer patients.Major advances have been made in CRC treatment in recent years, especially in molecularly driven therapies and immunotherapy. Despite this, a large number of advanced colorectal cancer patients do not benefit from these treatments and their prognosis remains poor. The landscape of DNA damage response (DDR) alterations is emerging as a novel target for treatment in different cancer types. PARP inhibitors have been approved for the treatment of ovarian, breast, pancreatic, and prostate cancers carrying deleterious BRCA1/2 pathogenic variants or homologous recombination repair (HRR) deficiency (HRD). Recent research reported on the emerging role of HRD in CRC and showed that alterations in these genes, either germline or somatic, are carried by up to 15–20% of CRCs. However, the role of HRD is still widely unknown, and few data about their clinical impact are available, especially in CRC patients. In this review, we report preclinical and clinical data currently available on DDR inhibitors in CRC. We also emphasize the predictive role of DDR mutations in response to platinum-based chemotherapy and the potential clinical role of DDR inhibitors. More preclinical and clinical trials are required to better understand the impact of DDR alterations in CRC patients and the therapeutic opportunities with novel DDR inhibitors.

Despite provocative preclinical results, dose-limiting toxicities have precluded rational combinations of cytotoxic chemotherapies that increase DNA damage with DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted delivery of cytotoxic chemotherapy might enable tolerable and active combinations with DDR inhibitors. We conducted a phase I clinical trial combining ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib with sacituzumab govitecan, a trophoblast cell surface antigen 2 (Trop-2) directed antibody drug conjugate (ADC) that delivers high tumoral concentrations of topoisomerase 1 (TOP1) inhibitor SN-38. Depletion of ATR, the main transducer of replication stress is synthetically lethal with double-strand breaks (DSB) generated by TOP1 inhibitors. Patients with DDR gene-mutated or high replication stress solid tumors were enrolled since such tumors are particularly susceptible to ATR inhibition. Primary end point was identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Using 3 + 3 dose escalation, sacituzumab govitecan (8-10 mg/m2, days 1, 8) and berzosertib (140-210 mg/m2, days 2, 9) were administered to 12 patients across three dose levels in 21-day cycles. The combination was well tolerated, with improved safety profile over conventional chemotherapy-based combinations, which allowed dose escalation to the highest planned dose level. There were no dose limiting toxicities. Common treatment-related adverse events (TRAE) were neutropenia (41.7%), diarrhea (50%), and fatigue (50%). Grade 3 TRAEs occurred in 58.3% of patients and included neutropenia (25%) and diarrhea (8.3%). There were no instances of febrile neutropenia or clinically significant grade 4 TRAEs. Pharmacodynamic studies showed evidence of ATR inhibition and enhanced DNA DSB in response to the combination. While no tumor responses were seen in three patients with DDR defects including BRCA1 and ATM mutations, two patients with neuroendocrine prostate cancer, a highly aggressive subtype of prostate cancer, showed partial or metabolic responses. A patient with EGFR-transformed small cell lung cancer (SCLC) also experienced partial response, together yielding objective responses in 3 of 12 evaluable patients (25%). Ongoing phase II expansion cohorts are evaluating efficacy of sacituzumab govitecan 10mg/m2 and berzosertib 210mg/m2 in patients with SCLC, extra-pulmonary small cell cancers, and DDR-mutated solid tumors. ADC-based delivery of cytotoxic payload represents a new therapeutic paradigm to extend the benefit of DDR inhibitors to target replication stress and chemotherapy resistance, with minimal added toxicities. Clinical trial information: NCT04826341 Citation Format: Melissa L. Abel, Nobukyuki Takahashi, Parth Desai, Cody Peer, Christophe Redon, Samantha Nichols, Rasa Vilimas, Min-Jung Lee, Sunmin Lee, Linda Sciuto, Danielle Pinkiert, Meenakshi Shelat, Chante Graham, Seth Steinberg, William D. Figg, Mirit Aladjem, Jane Trepel, Yves Pommier, Anish Thomas. Targeting replication stress and chemotherapy resistance with a combination of sacituzumab govitecan and berzosertib: A phase I clinical trial [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 CT268.

The serine/threonine kinase STK11 (LKB1) is the second most commonly altered tumor suppressor in NSCLC; however, there are currently no effective treatment strategies for this subset of tumors. KRAS-mutant LKB1 deficient tumors often also have alterations in KEAP1 or NRF2 gene, which activate the NRF2 pathway known to be involved in antioxidant response. Inhibitors of ATM and ATR, two key proteins in the DNA damage response (DDR) pathway, are currently undergoing clinical testing but there are no biomarkers established for identifying which subgroups of patients are more likely to benefit from treatment. Here we have identified that alterations of LKB1, and the KEAP1/NRF2 pathway, are associated with enhanced response to ATM and ATR inhibitors (AMTi and ATRi) and other inhibitors of the DDR and may be useful biomarkers for predicting therapeutic response. To investigate the impact of LKB1 loss and KEAP1/NRF2 pathway activation on response to DDR inhibitors (DDRi), we first tested the in vitro activity of ATM inhibitor in NSCLC murine cell lines with or without knock out of LKB1 and/or KEAP1. In these cells, the loss of LKB1 and/or KEAP1 significantly sensitize cells to ATMi AZD0156. In addition, we evaluated the activity of the ATRi AZD6738 in NSCLC cells with or without knockout of LKB1 and/or KEAP1. Cells deficient in LKB1 (KL) and/or KEAP1 (KLK/KK) were more sensitive to AZD0156 and AZD6738 than cells with intact LKB1 and KEAP1. Next, we investigated whether the activity of ATR and ATM inhibitors in KL, KK or KLK tumor cells could be enhanced by the addition of a PARP inhibitor (Olaparib). Although all NSCLC cells were resistant to the PARP inhibitor olaparib when used as a single agent, treatment of LKB1, KEAP1 or LKB1 plus KEAP1 deficient cells with the combination of olaparib plus ATM or ATR inhibitors significantly enhanced the antitumor cell activity of ATM or ATR inhibitors alone. We confirmed these data in an additional panel of LKB1 deficient NSCLC human cell lines (A549, H460 and H2030) treated with a broad spectrum of ATR and ATM inhibitors. In all human cell lines re-expression of LKB1 clearly reduced the sensitivity to ATR inhibition. LKB1 lost was also associated with sensitivity to PARP and ATM inhibitor, although these effects seemed to be less significant compared with ATR inhibitors. Tumors with LKB1 deficiency or KEAP/NRF2 mutations are typically resistant to standard chemotherapy drugs and immunotherapy. Our data indicate that LKB1 and KEAP1/NRF2 loss significantly enhance the sensitivity to ATR and ATM inhibitors in vitro. Thus, we have identified that NSCLC tumors bearing STK11 or KEAP1/NRF2 mutations are highly sensitive to ATM or ATR inhibitors and that genes may serve as biomarkers for selecting appropriate patients for treatment alone or in combination treatments, such as PARPi or immunotherapy. Citation Format: Ana Galan-Cobo, Alissa Pottetee, John V. Heymach. LKB1 deficiency and KEAP1/NRF2 pathway alterations as biomarkers of response for ATR and ATM inhibitors and other inhibitors of DNA damage response (DDR) in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3507.

Mutations in oncogenes, tumor suppressor and DNA damage response (DDR) mediator genes drive or permit malignant transformation but also increase endogenous replication stress. The serine/threonine kinase ATR plays a critical role in safeguarding genome integrity from such replication stress and several studies have demonstrated the increased reliance of cancer cells on ATR function. We investigated the therapeutic opportunities for the ATR inhibitor, AZD6738, in combination with DNA damaging or DDR-targeted agents, in the context of pancreatic ductal adenocarcinoma (PDAC). We evaluated four DNA-damaging agents (gemcitabine, 5-fluorouracil, oxaliplatin, SN38 (the active metabolite of irinotecan)) and three DDR-targeted agents (Wee1 inhibitor (AZD1775), Chk1 inhibitor (MK8776), PARP inhibitor (AZD2281)), each in combination with AZD6738 at multiple concentrations. Efficacy of these combinations was tested in growth inhibition assays in vitro, using a panel of cell lines in order to capture some of the genetic heterogeneity observed in PDAC: two human cell lines and four lines from the KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse. Synergistic growth inhibition was identified applying both Bliss Independence and Loewe models, using Combenefit software. All the KPC mouse cell lines were sensitive to AZD6738 as a single agent, with GI50 ranging from 346 to 566 nM. MIA PaCa-2 were sensitive to AZD6738, achieving >90% growth inhibition, with GI50 of 2.2 μM. PANC-1 cells were less sensitive, with GI50 21 μM and achieving only ~60% GI, at the highest concentration tested. PANC-1 cells are also less sensitive to gemcitabine than the other cell lines. Synergy was detected in most of the cell lines, with each of the seven drug combinations tested. The combinations of AZD6738 with gemcitabine and with oxaliplatin showed synergy in all cell lines tested. We next investigated scheduling of the gemcitabine/ATRi combination, at the specific GI50 concentrations for each cell line, using kinetic live-cell imaging assays. Concurrent treatment of gemcitabine/ATRi for 16h proved to be most effective, almost completely inhibiting cell growth for more than three days after washout. Sequential treatment (irrespective of the order) or shorter pulses (8h) were less effective. Maintaining ATRi after gemcitabine washout further enhanced growth inhibition for most cell lines. Mechanistically, ATRi impaired Chk1 activation (p-Ser345) and, in combination with gemcitabine, strongly potentiated DNA damage (gamma H2AX). Maintaining ATRi after gemcitabine washout helped to sustain the level of DNA damage. In vivo studies are underway to determine whether the gemcitabine/ATRi combination enhances efficacy compared to gemcitabine alone. The ATRi/oxaliplatin combination is also being investigated in vitro and in vivo using similar methods. Several genes have been described in the literature to increase the reliance on ATR functions when altered. Mining two published datasets (TCGA, 186 samples and UTSW, Nat. Commun. 2015, 109 samples) we have investigated the frequencies at which 21 of these genes were altered in human PDAC. Overall ~95% of PDAC samples exhibit at least one (9% only one, 28% two and 57% three or more) genetic alteration likely to sensitize to ATRi, potentially improving the therapeutic index of combination approaches. Thus, combinations including ATRi merit further evaluation as they have the potential to be effective in the treatment of patients with PDAC. Citation Format: Yann Wallez, Siang-Boon Koh, Venkata Sai Vivek Bhogadi, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The ATR inhibitor, AZD6738, synergizes with other DNA damage response inhibitors and genotoxic drugs in pancreatic ductal adenocarcinoma cell lines: Opportunities for new therapeutic combinations [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B19.

Poly(ADP-ribose) polymerase (PARP) inhibitors have transformed treatment paradigms in multiple cancer types defined by homologous recombination deficiency (HRD) and have become the archetypal example of synthetic lethal targeting within the DNA damage response (DDR). Despite this success, primary and acquired resistance to PARP inhibition inevitability threaten the efficacy and durability of response to these drugs. Beyond PARP inhibitors, recent advances in large-scale functional genomic screens have led to the identification of a steadily growing list of genetic dependencies across the DDR landscape. This has led to a wide array of novel synthetic lethal targets and corresponding inhibitors, which hold promise to widen the application of DDR inhibitors beyond HRD and potentially address PARP inhibitor resistance. In this review, we describe key synthetic lethal interactions that have been identified across the DDR landscape, summarize the early phase clinical development of the most promising DDR inhibitors, and highlight relevant combinations of DDR inhibitors with chemotherapy and other novel cancer therapies, which are anticipated to make an impact in rationally selected patient populations. The DDR landscape holds multiple opportunities for synthetic lethal targeting with multiple novel DDR inhibitors being evaluated on early phase clinical trials. Key challenges remain in optimizing the therapeutic window of ATR and WEE1 inhibitors as monotherapy and in combination approaches.

Background: High-grade serous ovarian carcinoma (HGSOC) is the most frequent histotype of ovarian cancer. More than half of HGSOC is characterized by homologous recombination deficiency (HRD) due to mutations in genes involved in this pathway, including BRCA1/2. Olaparib is a poly(ADP-ribose) polymerase inhibitor (PARPi) recently approved in front line and maintenance therapy in platinum-sensitive BRCA mutated ovarian cancer patients. Despite olaparib clinical benefits, this treatment is associated with the development of resistance. A better understanding of the mechanisms at its basis could help in finding strategies to delay or overcome/counteract it, possibly translatable in the clinical setting. Methods: We generated an olaparib resistant cell line (BRCA1-/- OlaR), starting from the BRCA1 and TP53 deleted murine ID8 cells (BRCA1-/-) by step wise increasing drug concentrations. Sensitive and resistant cell lines were biologically and pharmacologically characterized and the molecular mechanisms at the basis of olaparib resistance investigated. Cellular viability was assessed by MTS assay, for molecular studies real-time PCR, western blot and RAD51-foci immunofluorescence were performed. Results: BRCA1-/- OlaR cells were 24 fold time more resistant than BRCA1-/- cells and displayed a similar in vitro cell growth compared to the parental cells. Olaparib treatment at IC50 dose caused a longer and stronger G2-M block of the cell cycle in sensitive as compared to resistant cells. BRCA1-/- OlaR cells were cross-resistant to the other PARPis tested, suggesting a common mechanism of resistance. Platinum compounds showed similar cytotoxic activity in sensitive and resistant cells, while a partial cross-resistance was observed with doxorubicin. Interestingly, resistant cells were less responsive to a panel of DNA damage response (DDR) inhibitors (i.e. ATR, WEE1 and CHK1 inhibitors) compared to parental ones; however their combinations with olaparib were synergic in both the sensitive and resistant lines. A partial restoration of the HR pathway (increase in RAD51 foci formation after olaparib and IR treatment), a downregulation of PARP1 protein levels without mutation in PARP1 gene and an upregulation of the MDR transcript could be found in BRCA1-/- resistant cells, strongly supporting multiple heterogeneous mechanisms of resistance. Conclusions: The HR deficient cell line resistant to olaparib we generated showed multiple mechanisms of resistance. The combinations of olaparib with different DDR inhibitors were equally active in sensitive and resistant cells. Citation Format: Michela Chiappa, Martina Anselmi, Luca Russo, Monica Lupi, Nicolò Panini, Federica Guffanti, Giovanna Damia. Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells [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 3244.