PARP Inhibitor Resistance Research Articles

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 7146: HSK39775: A USP1 inhibitor for the treatment of cancers with homologous recombination deficiencies

Abstract Tumors with BRCA1/2 mutations or homologous repair deficiency (HRD) are vulnerable to agents that target DNA repair pathways, including platinum-containing chemotherapeutics and poly (ADP-ribose) polymerase-1 (PARP1) inhibitors. Despite the significant therapeutic benefits in patients with BRCAm or HRD, innate and acquired resistance to PARPi is still a challenge in the clinic. To address this clinical challenge, we developed HSK39775, a potent, highly selective inhibitor of ubiquitin carboxyl-terminal hydrolase 1 (USP1), which acts as an oncogenic and PARP inhibitor resistance driver involved in DNA damage repair pathway through the deubiquitylation of proteins in Translesion Synthesis pathway and Fanconi Anemia pathway, paired with BRCA as a synthetic lethality target. HSK39775 was active in enzymatic level to inhibit USP1/UAF1 complex. Enzymatic panel screening containing 50 deubiquitinase demonstrated high selectivity of HSK39775. Ubiquitinated PCNA was accumulated treated by HSK39775, indicating target engagement. HSK39775 also demonstrated potent anti-proliferation activity against cancer cell lines with BRCA mutation and excellent selectivity between BRCA WT vs. BRCAm. Combining HSK39775 with PARP inhibitors indicated synergy in cell lines with partial or insensitivity to each agent alone. Triple-negative BRCA mutant breast cancer xenograft model demonstrated strong dose-dependent tumor growth inhibition of HSK39775 as a single agent with Tumor Growth Inhibition (TGI) of 63.2% at 30 mg/kg QD, and ubiquitinated PCNA was also accumulated in HSK39775 treated tumor tissue. Meanwhile, combination with PARP inhibitors significantly enhanced the therapeutic efficacy of HSK39775 in xenograft model and led to tumor regressions and durable tumor control even at low dose (TGI:89% (HSK39775, 5 mg/kg + Olaparib, 50 mg/kg)). Interestingly, HSK39775 inhibited the tumor growth of a HRP and BRCA wild-type lung cancer xenograft as a single agent with TGI of 94% at 30 mg/kg QD, suggesting the therapeutic effect of USP1 inhibitors beyond canonical BRCA mutant or HRD tumors. In summary, these results indicate HSK39775 as a potent and selective USP1 inhibitor and support further development. Citation Format: Jun Xu, Yangguang Li, Ju Wang, Pingming Tang, Jianmin Wang, Chen Zhang, Pangke Yan. HSK39775: A USP1 inhibitor for the treatment of cancers with homologous recombination deficiencies [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 7146.

Abstract 4757: Combining PARP inhibitor with TACC3 inhibition overcomes PARP inhibitor resistance in ovarian cancer

Abstract The treatment of ovarian cancer (OC) presents a significant challenge due to the emergence of resistance to poly (ADP-ribose) polymerase inhibitors (PARPi). Transforming acidic coiled coil containing protein 3 (TACC3) belongs to the TACC family, characterized by its coiled-coil domains. TACC3 plays a crucial role in cell division, particularly in the formation and stability of the mitotic spindle, which is essential for proper chromosome segregation. Mutations or changes in TACC3 have been associated with various cancers, making it an increasingly attractive target for anticancer therapy in recent years. Here, we demonstrated a significant upregulation of TACC3 in OC, correlating with adverse clinical outcomes. Notably, PARPi-resistant OC cells exhibited a marked sensitivity to TACC3 inhibition. This was evidenced by the induction of spindle defects and mitotic catastrophe in PARPi-resistant cells upon TACC3 suppression, achieved either genetically or pharmacologically. Moreover, we explored the therapeutic efficacy of combining TACC3 inhibitor with PARPi, specifically BO-264 with olaparib or niraparib. This combination exhibited a synergistic effect, markedly reducing cell viability in vitro and tumor growth in PARPi-resistant tumor xenograft models. Our study suggests a promising new method for treating ovarian cancer, especially for those resistant to current PARPi treatments, by simultaneously targeting TACC3 and PARPi, enhancing treatment efficacy and potentially improving patient outcomes. Citation Format: Yu Wu, Qilong Wang, Chaolin Deng, Mo Li, Yinan Xiao. Combining PARP inhibitor with TACC3 inhibition overcomes PARP inhibitor resistance in ovarian 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 4757.

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.

Abstract B007: NAD+ metabolism drives PARPi resistance in ovarian cancer cells

Abstract NAD+ is a coenzyme for redox reactions and a cofactor for enzymes such as PARPs and Sirtuins. The nuclear enzyme PARP1 is a major consumer of NAD+, which is cleaved during the catalytic activation of PARP1 in response to DNA damage. PARP1 inhibitors compete with NAD+ for the catalytic site of PARP1 and reduce the catalytic activity of PARP1 and impair DNA damage repair. Recently PARP inhibitors (PARPi) have shown efficacy in the treatment of ovarian cancers and most patients with defects in homologous repair of DNA damage are now on PARPi maintenance. But, heterogeneous responses to PARPi is limiting their clinical efficacy. To identify the mechanisms of PARPi resistance, we generated a panel of ovarian cancer cells with acquired resistance to niraparib (PARPiR), an FDA approved PARP inhibitor. We demonstrated that the PARPiR cells are also resistant to other FDA approved PARP inhibitors rucaparib and olaparib. This data suggested that a common mechanism of resistance underlies poor response to the FDA approved PARP inhibitors. Since PARP1 activity is tightly controlled by the cellular NAD+ levels, we next investigated the role of NAD+ biosynthesis in PARPi resistance. Metabolomic profiling of the parental and PARPiR cells demonstrated that PARPiR cells have elevated levels of NAD+ metabolism. Indeed, treating the PARPiR cells with inhibitors of NAD+ synthesis restored the sensitivity to PARP inhibitors. Moreover, culturing the previously sensitive parental ovarian cancer cells in NAD+ precursors decreased their sensitivity to PARP inhibitors. These data collectively demonstrated that high levels of NAD+ metabolism causes PARP inhibitor resistance in ovarian cancer cells. Complementary to the metabolomic analysis, results from phenotypic assays in PARPiR cells revealed that high levels of NAD+ maintain cellular quiescence in PARPiR cells. In summary, our results demonstrated that high levels of NAD+ biosynthesis causes PARPi resistance by maintaining cellular quiescence. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. Our study identified that maintenance of quiescence is a fundamental mechanism of PARPi resistance. We also demonstrated that inhibiting NAD+ biosynthesis reverses PARP inhibitor resistance. Citation Format: Subin Myong, Hardik Shah, Benedikt Nagele, Reshma Rajesh, Sridevi Challa. NAD+ metabolism drives PARPi resistance in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B007.

Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2)is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. We surmised that alterations in metabolic pathways by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused clusteredregularly interspaced short palindromic repeats knockoutscreen to identify genes that undergo alterations during the treatment of tumor cells with PARPis. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing nonsmall cell lung cancer(NSCLC) lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple-negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the tricarboxylicacid cycle and oxidative phosphorylation pathways to overcome PARPi treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.

Discovery and Proof of Concept of Potent Dual Polθ/PARP Inhibitors for Efficient Treatment of Homologous Recombination-Deficient Tumors.

DNA polymerase theta (Polθ) has recently emerged as a new attractive synthetic lethal target involved in DNA damage repair. Inactivating Polθ alone or in combination with PARP inhibitors has demonstrated substantial therapeutic potential against tumors with homologous recombination (HR) defects such as alternation of BRCA genes. Herein, we report the design and proof of concept of a highly potent dual Polθ/PARP inhibitor 25d, which exhibited low nanomolar inhibitory activities against both Polθ and PARP1. Compared to combination treatment, 25d demonstrated superior antitumor efficacy in both MDA-MB-436 cells and xenografts by inducing more DNA damage and apoptosis. Importantly, 25d retained sensitivity in PARP inhibitor-resistant MDA-MB-436 cells with 53BP1 defect. Altogether, these findings illustrate the potential advantages of 25d, a first-in-class dual Polθ/PARP inhibitor, over monotherapy in treating HR-deficient tumors, including those with acquired PARP inhibitor resistance.

Reclassifying BRCA1 c.4358-2A > G and BRCA2 c.475 + 5G > C variants from “Uncertain Significance” to “Pathogenic” based on minigene assays and clinical evidence

BackgroundPathogenic variants in BRCA genes play a crucial role in the pathogenesis of ovarian cancer. Intronic variants of uncertain significance (VUS) may contribute to pathogenicity by affecting splicing. Currently, the significance of many intronic variants in BRCA has not been clarified, impacting patient treatment strategies and the management of familial cases.MethodA retrospective study was conducted to analyze BRCA intronic VUS in a cohort of 707 unrelated ovarian cancer patients at a single institution from 2018 to 2023. Three splicing predictors were employed to analyze detected intronic VUS. Variants predicted to have splicing alterations were selected for further validation through minigene assays. Patient and familial investigations were conducted to comprehend cancer incidence within pedigrees and the application of poly (ADP-ribose) polymerase inhibitors (PARPi) by the patients. In accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG), the intronic VUS were reclassified based on minigene assay results and clinical evidence.ResultApproximately 9.8% (69/707) of patients were identified as carriers of 67 different VUS in BRCA1/2, with four intronic variants accounting for 6% (4/67) of all VUS. Splicing predictors indicated potential splicing alterations in splicing for BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C variants. Minigene assays utilizing the pSPL3 exon trapping vector revealed that these variants induced changes in splicing sites and frameshift, resulting in premature termination of translation (p. Ala1453Glyfs and p. Pro143Glyfs). According to ACMG guidelines, BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C were reclassified as pathogenic variants. Pedigree investigations were conducted on patients with BRCA1 c.4358-2A>G variant, and the detailed utilization of PARPi provided valuable insights into research on PARPi resistance.ConclusionTwo intronic VUS were reclassified as pathogenic variants. A precise classification of variants is crucial for the effective treatment and management of both patients and healthy carriers.

Abstract B032: USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells

Abstract Targeting BRCA-deficient tumors with PARP inhibitors has improved the progression-free survival of ovarian and breast cancer patients in the clinic. However, the majority of patients develop acquired resistance. Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulating single-stranded DNA (ssDNA) gaps at replication forks. We have previously shown that USP1 deficiency is synthetic lethal with BRCA1 deficiency, suggesting USP1 is a potential therapeutic target for overcoming PARPi resistance (Lim et al., Mol Cell, 2018). Accordingly, USP1 inhibitors (USPi) are now undergoing clinical development. Here, we show that USP1i also promote ssDNA gap accumulation during replication in BRCA1-deficient cells. USP1 inhibition results in the accumulation of monoubiquitinated PCNA at the replication fork and increased levels of non-cleaved USP1 and RAD18 at the replication fork. Accumulation of ssDNA gaps is dependent on RAD18 activity and monoubiquitinated PCNA levels. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, resulted in reduced PCNA-Ub levels, resistance to the USP1i, and suppression of ssDNA gaps. Moreover, the accumulation of ssDNA gaps is associated with increased S-phase PAR levels and is independent of PRIMPOL, suggesting defects on the lagging strand of the replication fork. Interestingly, the induction of ssDNA gaps was detected in USP1i-sensitive but not USP1i-resistant BRCA1-deficient cells, suggesting this is the mechanism of cytotoxicity by USP1 inhibition. Indeed, USP1i also sensitized BRCA1-deficient cancer cells to other ssDNA gap-inducing agents such as POLQ and PARP inhibitors. Notably, USP1i exhibited monotherapy activity in ovarian and breast cancer cell lines with acquired PARPi resistance and re-sensitized them to PARP inhibition. PARPi-resistant BRCA1-mutated patient-derived ovarian cancer organoids were also sensitized by USP1 inhibition. Similar to the cell lines, the ssDNA gap accumulation induced by USP1i in organoids strongly correlated with the drug sensitivity of both PARPi-sensitive and PARPi-resistant models. Finally, USPi overcame PARPi resistance in a BRCA1-mutant PARPi-resistant PDX model. In conclusion, USPi exhibits monotherapy activity in both PARPi-sensitive. Citation Format: Alexandre A. B. A. da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David B. Martignetti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey I. Shapiro, Kalindi Parmar, Alan D. D’Andrea. USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B032.

Abstract IA001: Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity

Abstract Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA gaps (ssDNA gaps) at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells. USP1 inhibition increased monoubiquitinated PCNA at replication forks and resulted in accumulation of ssDNA gaps in BRCA1-deficient cancer cells. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, caused USP1 inhibitor resistance and suppression of ssDNA gaps. ssDNA gap accumulation induced by USP1 inhibition correlates with drug sensitivity of BRCA1-mutated cancer cells and overcomes PARP inhibitor resistance in cell line and xenograft models. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. A set of patient-derived ovarian cancer organoids (PDOs) were used to confirm the sensitivity of BRCA1-deficient cells to USP1 inhibition. Five PDOs were derived from patients with high grade-serous ovarian cancer (HGSOC). Among the five PDOs, two of them were from tumors harboring germline BRCA1 pathogenic mutations. BRCA1-mutant PDOs, but not BRCA1-WT PDOs, were sensitive to a USP1 inhibitor and to PARP inhibitor monotherapies. The accumulation of ssDNA gaps after treatment with a USP1 inhibitor or PARP inhibitor correlated with the sensitivity to these drugs in all the models tested. Moreover, the combination of a PARP inhibitor and USP1 inhibitor showed synergy in a BRCA1-WT model, which was resistant to both monotherapies. Interestingly, ssDNA gaps accumulated with the combination treatment and not with monotherapy in this model. Ovarian cancer PDOs therefore provide a powerful tool for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to USP1 inhibition as monotherapy or in combination in ongoing clinical trials. Citation Format: Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA001.

Abstract A015: 53BP1 loss mediated PARP inhibitor resistance in BRCA1-deficient pancreatic cancer is overcome with immune checkpoint blockade

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer characterized by the presence of mutations in homologous recombination (HR) repair genes, including BRCA1 and BRCA2, in approximately 20% of diagnosed cases. 53BP1 (tumor protein p53 binding protein 1) nucleates the anti-end resection machinery at DNA double strand break (DSB)s thereby countering BRCA1 activity. Importantly, BRCA1-deficient PDACs that are typically sensitive to poly-ADP ribose polymerase inhibitors (PARPi) become resistant in the absence of 53BP1. Here, we demonstrate that loss of 53BP1 results in enhanced DNA end-resection, increased micronuclei, and cytoplasmic dsDNA, leading to activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, activating pro-inflammatory signaling. This not only enhances CD8+ T cell infiltration but also activates macrophages and natural killer (NK) cells culminating in tumor growth inhibition. Loss of 53BP1 correlates with response to immune checkpoint blockade (ICB) and improved overall survival. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Taken together, our findings provide a rationale for combining PARPi with ICB to overcome PARPi-resistance in BRCA1-deficient pancreatic cancers. Citation Format: Jeffrey Patterson-Fortin, Yajie Sun, Sen Han, Zhe Li, Zuzanna Nowicka, Yuna Hirohashi, Susan Kilgas, Jae Kyo Yi, Alexander Spektor, Wojciech Fendler, Panagiotis Konstantinopoulos, Alan D. D'Andrea, Dipanjan Chowdhury. 53BP1 loss mediated PARP inhibitor resistance in BRCA1-deficient pancreatic cancer is overcome with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A015.

PLK1 and PARP positively correlate in Middle Eastern breast cancer and their combined inhibition overcomes PARP inhibitor resistance in triple negative breast cancer.

Despite advancements in treatment approaches, patients diagnosed with aggressive breast cancer (BC) subtypes typically face an unfavorable prognosis. Globally, these cancers continue to pose a significant threat to women's health, leading to substantial morbidity and mortality. Consequently, there has been a significant struggle to identify viable molecular targets for therapeutic intervention in these patients. Polo-like Kinase-1 (PLK1) represents one of these molecular targets currently undergoing rigorous scrutiny for the treatment of such tumors. Yet, its role in the pathogenesis of BC in Middle Eastern ethnicity remains unexplored. We investigated the expression of PLK1 protein in a cohort of more than 1500 Middle Eastern ethnicity BC cases by immunohistochemistry. Association with clinicopathological parameters and prognosis were performed. In vitro studies were conducted using the PLK1 inhibitor volasertib and the PARP inhibitor olaparib, either alone or in combination, in PTC cell lines. Overexpression of PLK1 was detected in 27.4% of all BC cases, and this was notably correlated with aggressive clinicopathological markers. PLK1 was enriched in the triple-negative breast cancer (TNBC) subtype and exhibited poor overall survival (p = 0.0347). Notably, there was a positive correlation between PLK1 and PARP overexpression, with co-expression of PLK1 and PARP observed in 15.7% of cases and was associated with significantly poorer overall survival (OS) compared to the overexpression of either protein alone (p = 0.0050). In vitro, we studied the effect of PLK1 and PARP inhibitors either single or combined treatments in two BRCA mutated, and one BRCA proficient TNBC cell lines. We showed that combined inhibition significantly reduced cell survival and persuaded apoptosis in TNBC cell lines. Moreover, our findings indicate that inhibition of PLK1 can reinstate sensitivity in PARP inhibitor (PARPi) resistant TNBC cell lines. Our results shed light on the role of PLK1 in the pathogenesis and prognosis of Middle Eastern BC and support the potential clinical development of combined inhibition of PLK1 and PARP, a strategy that could potentially broaden the use of PLK1 and PARP inhibitors beyond BC cases lacking BRCA.

Paris saponin VII reverses resistance to PARP inhibitors by regulating ovarian cancer tumor angiogenesis and glycolysis through the RORα/ECM1/VEGFR2 signaling axis.

The emergence of Poly (ADP-ribose) polymerase inhibitors (PARPi) has marked the beginning of a precise targeted therapy era for ovarian cancer. However, an increasing number of patients are experiencing primary or acquired resistance to PARPi, severely limiting its clinical application. Deciphering the underlying mechanisms of PARPi resistance and discovering new therapeutic targets is an urgent and critical issue to address. In this study, we observed a close correlation between glycolysis, tumor angiogenesis, and PARPi resistance in ovarian cancer. Furthermore, we discovered that the natural compound Paris saponin VII (PS VII) partially reversed PARPi resistance in ovarian cancer and demonstrated synergistic therapeutic effects when combined with PARPi. Additionally, we found that PS VII potentially hindered glycolysis and angiogenesis in PARPi-resistant ovarian cancer cells by binding and stabilizing the expression of RORα, thus further inhibiting ECM1 and interfering with the VEGFR2/FAK/AKT/GSK3β signaling pathway. Our research provides new targeted treatment for clinical ovarian cancer therapy and brings new hope to patients with PARPi-resistant ovarian cancer, effectively expanding the application of PARPi in clinical treatment.

TH‐302: A Highly Selective Hypoxia‐Activated Prodrug for Treating PARP Inhibitor–Resistant Cancers

Introduction: Poly (ADP‐ribose) polymerase (PARP) inhibitor has been widely used in ovarian cancer patients carrying BRCA mutations. However, resistance to PARP inhibitor is present in some patients, and no effective treatment is available for these patients. TH‐302 is a hypoxia‐activated prodrug, which releases the bis‐DNA alkylator bromo‐isophosphoramide mustard (Br‐IPM) under hypoxic condition. The present study aims to determine whether TH‐302 is effective in treating PARP inhibitor resistance.Methods: The in vitro cytotoxicity of TH‐302 was assessed by short‐term proliferation assay (50% inhibitory concentration, IC50) or long‐term clonogenic assay (90% inhibitory concentration, IC90) under various oxygen concentrations. In vivo efficacy of TH‐302 was assessed in PARP inhibitor resistance, partially responsive and sensitive patient‐derived xenograft (PDX) or cell line–derived xenograft (CDX) models. Antitumor activity via homologous recombination (HR) pathway for TH‐302 was evaluated using DLD1 BRCA2 knockout cell line and BRCA/RAD51D deleterious mutant PDX/CDX models. Breaks of double‐strand DNA and hypoxia fraction in tumors were determined by gamma histone 2AX (γH2AX) and pimonidazole immunohistochemistry in H460 CDX model following treatment.Results: Cytotoxicity was significantly enhanced under hypoxia in 12 human cancer cell lines including four ovarian cancer cell lines. The cytotoxicity was 70 times higher in human colon cancer cell line with BRCA2 knock out compared to wild type under hypoxia following TH‐302 treatment. γH2AX staining revealed that the cytotoxicity of TH‐302 was associated with DNA damage. In addition, administration of TH‐302 with olaparib led to better antitumor activities than either single drug/prodrug in olaparib‐resistant PDX models.Conclusion: TH‐302 exhibits hypoxia‐dependent cytotoxicity across a wide range of human cancer cell lines, and may be a drug candidate to treat ovarian cancer, bladder cancer, and pancreatic cancer with HR deficiencies with or without resistance to PARP inhibitor. TH‐302 may be effective in recurrent epithelial ovarian cancer (EOC) with homologous recombination deficiency (HRD) or in EOC patients resistant to PARP inhibitors.

Poly (ADP-ribose) polymerase inhibitor therapy and mechanisms of resistance in epithelial ovarian cancer.

Advanced epithelial ovarian cancer is the commonest cause of gynaecological cancer deaths. First-line treatment for advanced disease includes a combination of platinum-taxane chemotherapy (post-operatively or peri-operatively) and maximal debulking surgery whenever feasible. Initial response rate to chemotherapy is high (up to 80%) but most patients will develop recurrence (approximately 70-90%) and succumb to the disease. Recently, poly-ADP-ribose polymerase (PARP) inhibition (by drugs such as Olaparib, Niraparib or Rucaparib) directed synthetic lethality approach in BRCA germline mutant or platinum sensitive disease has generated real hope for patients. PARP inhibitor (PARPi) maintenance therapy can prolong survival but therapeutic response is not sustained due to intrinsic or acquired secondary resistance to PARPi therapy. Reversion of BRCA1/2 mutation can lead to clinical PARPi resistance in BRCA-germline mutated ovarian cancer. However, in the more common platinum sensitive sporadic HGSOC, the clinical mechanisms of development of PARPi resistance remains to be defined. Here we provide a comprehensive review of the current status of PARPi and the mechanisms of resistance to therapy.

Focus on Trabectedin in Ovarian Cancer: What Do We Still Need to Know?

In the era of single and combination maintenance therapies as well as platinum and Poly (ADP-ribose) polymerase inhibitors (PARPi) resistance, the choice of subsequent treatments following first-line platinum-based chemotherapy in recurrent ovarian cancer (ROC) patients has become increasingly complex. Within the ovarian cancer treatment algorithm, particularly in the emerging context of PARPi resistance, the role of trabectedin, in combination with pegylated liposomal doxorubicin (PLD) still preserves its significance. This paper offers valuable insights into the multifaceted role and mechanism of action of trabectedin in ROC. The main results of clinical trials and studies involving trabectedin/PLD, along with hints of Breast Cancer genes (BRCA)-mutated and BRCAness phenotype cases, are critically discussed. Moreover, this review provides and contextualizes potential scenarios of administering trabectedin in combination with PLD in ROC, according to established guidelines and beyond.

Unravelling the molecular basis of PARP inhibitor resistance in prostate cancer with homologous recombination repair deficiency.

Prostate cancer is a disease with heterogeneous characteristics, making its treatability and curability dependent on the cancer's stage. While prostate cancer is often indolent, some cases can be aggressive and evolve into metastatic castration-resistant prostate cancer (mCRPC), which is lethal. A significant subset of individuals with mCRPC exhibit germline and somatic variants in components of the DNA damage repair (DDR) pathway. Recently, PARP inhibitors (PARPi) have shown promise in treating mCRPC patients who carry deleterious alterations in BRCA2 and 13 other DDR genes that are important for the homologous recombination repair (HRR) pathway. These inhibitors function by trapping PARP, resulting in impaired PARP activity and increased DNA damage, ultimately leading to cell death through synthetic lethality. However, the response to these inhibitors only lasts for 3-4 months, after which the cancer becomesPARPi resistant. Cancer cells can develop resistance to PARPi through numerous mechanisms, such as secondary reversion mutations in DNA repair pathway genes, heightened drug efflux, loss of PARP expression, HRR reactivation, replication fork stability, and upregulation of Wnt/Catenin and ABCB1 pathways. OvercomingPARPi resistance is a critical and complex process, and there are two possible ways to sensitize the resistance. The first approach is to potentiate thePARPi agents through chemo/radiotherapy and combination therapy, while the second approach entails targeting different signaling pathways. This review article highlights the latest evidence on the resistance mechanism ofPARPi in lethal prostate cancer and discusses additional therapeutic opportunities available for prostate cancer patients with DDR gene alterations who do not respond toPARPi.

ATF3-induced activation of NF-κB pathway results in acquired PARP inhibitor resistance in pancreatic adenocarcinoma.

Olaparib, an inhibitor of poly-(adenosine diphosphate-ribose) polymerase (PARP), has been shown to have anticancer benefits in patients with pancreatic cancer who have a germline mutation in BRCA1/2. However, resistance acquired on long-term exposure to olaparib significantly impedes clinical efficacy. In this study, the chromatin accessibility and differentially expressed transcripts of parental and olaparib-resistant pancreatic cancer cell lines were assessed using the Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) and mRNA-seq. Detection of downstream genes regulated by transcription factors using ChIP (Chromatin immunoprecipitation assay). According to pathway enrichment analysis, differentially expressed genes in olaparib-resistant cells were remarkably enriched in the NF-κB signaling pathway. With ATAC-seq, we identified chromatin regions with higher accessibility in olaparib-resistant cells and predicted a series of important transcription factors. Among them, activating transcription factor 3 (ATF3) was significantly highly expressed. Functional experiments verified that inhibition of ATF3 suppressed the NF-κB pathway significantly and restored olaparib sensitivity in olaparib-resistant cells. Experiments in vitro and in vivo indicate ATF3 enhances olaparib resistance through the NF-κB signaling pathway, suggesting that ATF3 could be employed as an olaparib sensitivity and prognostic indicator in patients with pancreatic cancer.

Abstract B061: Sensitivity and resistance mechanisms of human cancer cell lines treated with PARP, POLθ, and ATR inhibitor combinations in 2D and 3D spheroid cell viability assays

Abstract Poly ADP Ribose Polymerase (PARP) inhibitors have changed the landscape of treatment of homologous recombination (HR) -deficient cancers. The combination of PARP targeting agents and genetic alterations in the HR pathway is known to be synthetically lethal. Currently, four PARP inhibitors are approved for treatment of HR-deficient cancers. In combination with DNA damaging agents, PARP inhibitors synergistically kill tumor cells. Occurrence of resistance to PARP inhibitors is a major problem in the clinic. Targeting alternative DNA repair pathways or the use of new drug combinations may overcome PARP inhibitor resistance. We have determined and compared the effect of inhibitors of PARP, POLθ and ATR on cell viability in a large panel of human cancer cell lines (the Oncolines® panel) in 2D cell viability assays, using intracellular ATP content as an indirect read-out of cell number. The relationship between the mutation and expression status of genes involved in the DNA damage response pathway, or known to be involved in PARP inhibitor resistance, was determined. The effect of co-treatment of PARP inhibitors with inhibitors of POLθ, ATR and DNA damaging, cytotoxic agents was determined in BLISS combination matrix experiments in the colorectal adenocarcinoma cell line DLD1 and an isogenic BRCA2-deficient knock-out derivative. Drug combinations were studied in 2D and in 3D spheroid viability assays. To identify novel, potential mechanisms of resistance to PARP inhibitors, BRCA1-deficient ovarian UWB1.289 and BRCA2-deficient DLD1 cells were treated with sequentially increasing concentrations of PARP inhibitors talazoparib or olaparib. Resistant clones were selected and characterized. Our study reveals insight into the genetic determinants of cancer cell line sensitivity to inhibitors of PARP, POLθ and ATR. We also present the first side-by-side comparative study of the effect of drug combinations targeting the DNA damage response pathway in HR-deficient tumors in 2D and 3D spheroid cell viability assays. Citation Format: Janneke J.T.M. Melis, Jeffrey Kooijman, Sabine Wetemans, Suzan Geerdink-Datema, Jelle Dylus, Jeroen A.D.M. de Roos, Wilhelmina E. van Riel, Guido J.R. Zaman. Sensitivity and resistance mechanisms of human cancer cell lines treated with PARP, POLθ, and ATR inhibitor combinations in 2D and 3D spheroid cell viability assays [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 B061.

Abstract B058: Combination of the PARP1-selective inhibitor AZD5305 with the ATR inhibitor ceralasertib for the treatment of PARPi-resistant cancer

Abstract AZD5305 is a novel, selective inhibitor of PARP1 and ceralasertib (AZD6738) an inhibitor of the ATR kinase, that target distinct DNA damage response pathways. Trapping of PARP1 increases replication stress and activates ATR. Combination of PARP and ATR inhibitors enhances anti-tumour efficacy and can overcome acquired PARP inhibitor resistance. The combination of the first-in-class PARP1 selective inhibitor AZD5305 with ceralasertib was explored in preclinical models of multiple tumour types, investigating dose and schedules to optimise efficacy while minimising exposure to mitigate potential tolerability issues. AZD5305 and ceralasertib combinations were evaluated in a cell panel screen and were more effective than the respective single agents in 37/114 (~32%) cancer cell lines, including (but not restricted to) homologous recombination repair-proficient lines. Breast cancer models that have wild type BRCA, and BRCA1 hypomorphs (that re-express the BRCA1 coding sequence), were sensitive to the combination of AZD5305 + ceralasertib. Long-term 28-day in vitro colony regression assays in PARP- and ATR inhibitor resistant cell lines, were used to determine the minimum-maximum clinically relevant drug concentrations required to drive cell kill. Drug washouts were carried out to evaluate various schedules of administration. In these experiments, low concentrations (6 nM) AZD5305 were sufficient to drive combination benefit and colony regressions. Sequence-scheduling evaluation indicated that concurrent administration and 7-day exposures at the start of the cycle, were sufficient for strong combination activity. 7-day AZD5305 exposures above 6 nM were sufficient for maximum activity. Higher concentrations or longer duration of treatment provided little additional benefit. In vivo efficacy of the combination was evaluated in PARPi-resistant models of breast and ovarian cancer. Dosing 0.1 mg/kg AZD5305, with ceralasertib for 14-days in a 28-day cycle achieved maximum anti-tumour efficacy. PDX models of TNBC or ER+ breast cancer that were resistant to monotherapies achieved complete regressions after combination dosing. These data establish low-dose, short-course concurrent dosing of AZD5305 in combination with ceralasertib is sufficient for maximal efficacy in PARP inhibitor resistant models. The combination of AZD5305 and ceralasertib shows activity in a range of PARPi- and ATRi-resistant tumor models. A clinical study of the combination of AZD5305 and ceralasertib is ongoing (NCT02264678). Citation Format: Mark Albertella, Paul Wijnhoven, Annie Demin, Giuditta Illuzzi, Andrea Herencia Ropero, Violeta Serra, Ankur Karmokar, Anna Staniszewska, Alan Lau. Combination of the PARP1-selective inhibitor AZD5305 with the ATR inhibitor ceralasertib for the treatment of PARPi-resistant cancer [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 B058.