Mechanisms Of PARP Inhibitor Resistance Research Articles

Activated NAD+ biosynthesis pathway induces olaparib resistance in BRCA1 knockout pancreatic cancer cells.

PARP inhibitors have been developed as anti-cancer agents based on synthetic lethality in homologous recombination deficient cancer cells. However, resistance to PARP inhibitors such as olaparib remains a problem in clinical use, and the mechanisms of resistance are not fully understood. To investigate mechanisms of PARP inhibitor resistance, we established a BRCA1 knockout clone derived from the pancreatic cancer MIA PaCa-2 cells, which we termed C1 cells, and subsequently isolated an olaparib-resistant C1/OLA cells. We then performed RNA-sequencing and pathway analysis on olaparib-treated C1 and C1/OLA cells. Our results revealed activation of cell signaling pathway related to NAD+ metabolism in the olaparib-resistant C1/OLA cells, with increased expression of genes encoding the NAD+ biosynthetic enzymes NAMPT and NMNAT2. Moreover, intracellular NAD+ levels were significantly higher in C1/OLA cells than in the non-olaparib-resistant C1 cells. Upregulation of intracellular NAD+ levels by the addition of nicotinamide also induced resistance to olaparib and talazoparib in C1 cells. Taken together, our findings suggest that upregulation of intracellular NAD+ is one of the factors underlying the acquisition of PARP inhibitor resistance.

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 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.

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.

RAD51D Secondary Mutation-Mediated Resistance to PARP-Inhibitor-Based Therapy in HGSOC.

Ovarian cancer is the leading cause of gynecologic cancer-related death, and PARP inhibitors (PARPis) are becoming a promising treatment option, as demonstrated by recent clinical trials. After PARPi exposure, somatic reversion mutations in the homologous recombination genes may be a mechanism of PARPi resistance in ovarian carcinoma. We present an ovarian cancer case of a 61-year-old woman, who underwent routine tumor reduction surgery followed by platinum and PARPis. She demonstrated a good response to PARPis for 15 months before recurrence and secondary tumor reduction surgery. However, post-surgery platinum and PARPi treatment only kept the disease stable for 5 months. A potential molecular mechanism for PARPi resistance was investigated using next-generation sequencing, immunohistochemical (IHC) staining, and other functional assays. A germline RAD51D loss-of-function mutation was found in the reported case (LRG_516t1:c.270_271dup p1:p.(Lys91fs*13)). Subsequently, a secondary mutation (LRG_516t1:c.271_282 del) was identified in the same locus of the germline duplication in the post-progression biopsies and ctDNA. The IHC staining supported low expression of RAD51D in the initial tumor tissue, but the expression was restored after the correction of the open reading frame by the secondary mutation. The in vitro results supported that the loss-of-function mutation of RAD51D was the basis for the initial response to the platinum and PARPi therapy, while the newly acquired reversion mutation could be attributed to the observed PARPi resistance. An acquired mutation can reverse a loss-of-function change in RAD51D and can result in PARPi resistance in a hereditary ovarian cancer patient. Liquid biopsy could be considered for longitudinal monitoring in ovarian patients under PARPi-based therapy, which can identify acquired resistant mutations earlier and facilitate precision management.

Treatment of Ovarian Cancer Beyond PARP Inhibition: Current and Future Options.

Ovarian cancer is the leading cause of gynecological cancer death. Improved understanding of the biologic pathways and introduction of poly (ADP-ribose) polymerase inhibitors (PARPi) during the last decade have changed the treatment landscape. This has improved outcomes, but unfortunately half the women with ovarian cancer still succumb to the disease within 5years of diagnosis. Pathways of resistance to PARPi and chemotherapy have been studied extensively, but there is an unmet need to overcome treatment failure and improve outcome. Major mechanisms of PARPi resistance include restoration of homologous recombination repair activity, alteration of PARP function, stabilization of the replication fork, drug efflux, and activation of alternate pathways. These resistant mechanisms can be targeted to sensitize the resistant ovarian cancer cells either by rechallenging with PARPi, overcoming resistance mechanism or bypassing resistance pathways. Augmenting the PARPi activity by combining it with other targets in the DNA damage response pathway, antiangiogenic agents and immune checkpoint inhibitors can potentially overcome the resistance mechanisms. Methods to bypass resistance include targeting non-cross-resistant pathways acting independent of homologous recombination repair(HRR), modulating tumour microenvironment, and enhancing drug delivery systems such as antibody drug conjugates. In this review, we will discuss the first-line management of ovarian cancer, resistance mechanisms and potential strategies to overcome these.

Update on poly(ADP-ribose) polymerase inhibitors resistance in ovarian cancer.

Ovarian cancer is one of the most common reproductive system tumors. The incidence of ovarian cancer in China is on the rise. Poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) is a DNA repair enzyme associated with DNA damage repair. PARPi takes PARP as a target to kill tumor cells, especially for tumors with homologous recombination (HR) dysfunction. Currently, PARPi has been widely used in clinical practice, mainly for the maintenance of advanced ovarian epithelial cancer. The intrinsic or acquired drug resistance of PARPi has gradually become an important clinical problem with the wide application of PARPi. This review summarizes the mechanisms of PARPi resistance and the current progress on PARPi-based combination strategies.

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanismsthat do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance invivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.

Abstract 870: Glutamine metabolism drives some mechanisms of PARP inhibitor persistence in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer with a five-year survival rate of 49.7%. Almost 1 in 6 patients dies within the first three months of diagnosis. Targeted therapies such as poly (ADP-ribose) polymerase (PARP) inhibitors have gained popularity due to their reduced toxicities. However, only 41% of patients respond to PARP inhibitors and most patients develop resistance. Here, we explored mechanisms of PARP inhibitor resistance in ovarian cancer at single-cell resolution to characterize potentially transient transcriptomic phenotypes associated with resistance. Parental (sensitive) and PARP inhibitor-persistent populations from two ovarian cancer cell lines (OVCAR4 and OVCA3) were sequenced by single-cell RNA sequencing (10X Genomics). Count matrices were log-normalized and scaled to remove variation due to read counts. Dimension reduction was performed using principal component analysis (PCA); cells were clustered phenotypically with graph-based clustering (Louvian algorithm) and visualized with UMAP projections. Downstream analysis includes characterization of the cell cycle phase, calculation of single-cell metabolic activity scores across 37 KEGG metabolic pathways, and pseudotime evolutionary trajectory analysis was performed to study the transition from the sensitive to the persistent phenotype. Across sensitive populations, most cells were in G1-phase; however, after developing persistence to PARP inhibitors, the OVCAR4 cells arrested at G2M phase, while the OVCA3 cells continued to arrest in the G1 phase. Our metabolic single cell RNA seq analysis pipelines revealed that OVCA3 sensitive population expressed high glycolysis metabolic scores compared to their persistent population, while the OVCAR4 persistent population showed a shift toward oxidative phosphorylation compared to the sensitive population implying the heterogenous metabolic adaptation across cancer cells. Persistent cells have high glutamine metabolism scores, suggesting that this pathway is upregulated in these populations compared to the parental ancestors. These findings were experimentally validated in cell lines treated with glutamine deprived media treated with different concentrations of PARP inhibitor. We used Palantir to characterize the evolutionary trajectories of the cells based on predictions of cell fate probabilities. OVCAR4 cells were predicted to develop into two distinct transcriptomic phenotypes, which could be related with sensitivity or tolerance to PARP inhibitors. However, OVCA3 cell did not have distinct, clear differentiation trajectories suggesting that transcriptomic information alone is not sufficient to explain the development of resistance in this cell line. These approaches further our understanding PARP inhibitor resistance in ovarian cancer, which is needed to improve current treatment strategies. Citation Format: Adriana Del Pino Herrera, Meghan Ferrall-Fairbanks, Mehdi Damaghi, Joon-Hyun Song, Snigdha Kanadibhotla. Glutamine metabolism drives some mechanisms of PARP inhibitor persistence in ovarian cancer [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 870.

Abstract 416: Phosphorylation of CDK9 transduces ALK signaling to promote resistance to PARP inhibitors via RNA Pol II activated homologous recombination repair

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are currently the most promising drugs to induce synthetic lethality in tumor with homologous recombination (HR) deficiency. However, development of resistance to PARPi is an almost universal occurrence in tumor despite initial sensitivity. Additionally, how to identify the right patients for PARPi monotherapy or in combination with other agents remains a substantial clinical challenge. Therefore, understanding the molecular underpinnings of PARPi resistance is critical to the development of rational combinatorial treatment strategies and identification of novel biomarkers which will in turn facilitate optimization of outcomes for patients receiving PARPi. By employing a screening procedure using phospho-RTK antibody array, our study identifies phosphorylated ALK (p-ALK) expression is higher in PARPi/platinum resistant cells relative to sensitive cells. We further demonstrate that p-ALK expression in human ovarian cancer tissue is associated with resistance to PARPi and platinum-based chemotherapy. Notably, FDA approved ALK inhibitors induce an HR-deficient phenotype and sensitize PARPi/platinum-resistant tumors to PARPi in vitro and in vivo. Mechanistically, ALK contributes to PARPi resistance by tyrosine phosphorylating CDK9 at Y19, thereby increasing the protein stability and kinase activity of CDK9 which in turn stabilizes positive transcriptional elongation factor b (P-TEFb) to activate RNA pol II and turn on HR repair genes transcription. Importantly, the phosphorylated counterparts of ALK/CDK9 complex (p-ALK/p-Y19 CDK9) can serve as a biomarker to identify patients with PARPi/platinum-resistant tumors which most likely to respond to dual PARP and ALK inhibition. Together, our findings identify p-ALK/p-Y19-CDK9 mediated stabilization of P-TEFb as a critical mechanism of PARPi resistance and supports a biomarker-guided therapeutic strategy combining ALKi and PARPi to induce synthetic lethality. Citation Format: Yu-Yi Chu, Jianjun Gao, Dihua Yu, Mien-Chie Hung. Phosphorylation of CDK9 transduces ALK signaling to promote resistance to PARP inhibitors via RNA Pol II activated homologous recombination repair [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 416.

Abstract ED12-1: Pol-theta inhibitors in HRD tumors

Abstract Deficiency of homologous recombination (HR)-mediated DNA repair occurs through genetic or epigenetic inactivation of the BRCA1 and BRCA2 (BRCA1/2) genes, and it plays a role in the initiation and progression of many tumor types. HR-deficiency also provides unique opportunities for targeted therapy, as exemplified by the extreme sensitivity of BRCA1/2-mutated tumors to poly (ADP-ribose) polymerase inhibitors (PARPi). In the last decade, several PARP inhibitors have been approved for clinical use. At first, PARPi were solely used in combination with chemotherapeutics, and platinum sensitivity was used as a surrogate marker of HR-deficiency for enrolling patients for treatment. The promising clinical response of patients with germline BRCA1/2-mutations prompted the use of PARPi for patients with somatic BRCA1/2 mutations as well. In addition, it also opened the possibility for extended use of PARPi for the treatment of various types of ovarian, breast, pancreatic and prostate tumors with HR defects. PARPi efficacy is currently being evaluated in different clinical settings such as first line chemotherapy, neoadjuvant therapy, and combination therapy with chemo- or immuno-therapies. Despite initial sensitivity to PARPi, resistance to these drugs is emerging as the major obstacle to its clinical effectiveness in patients with HR-deficient tumors. PARPi resistance can result from several independent mechanisms, often leading to the restoration of Homologous Recombination and/or Replication Fork stabilization. In addition, resistance to PARPi often correlates with platinum resistance, which remains the backbone therapy for most BRCA1/2-mutated tumors. The absence of alternative therapeutic options for patients with tumors with innate or acquired resistance underlines the urgency to develop additional therapeutics. While several mechanisms of PARPi resistance have been described, an effective method for overcoming such resistance is still lacking. DNA polymerase theta (POLθ or POLQ) recently emerged as a new promising drug target for the treatment of HR-deficient tumors. POLθ expression is particularly high in subtypes of breast and ovarian tumors with defects in HR, where it mediates backup DNA double-strand breaks (DSBs) repair, thus compensating for the loss of HR. As a result, POLθ is synthetic lethal with HR, and POLθ inhibition in HR-deficient tumors induces cell death. In addition, POLθ inhibition synergizes with PARPi in the killing of HR-deficient tumors. Synthetic lethality between HR-deficiency and POLθ inhibition hinges on several functions of POLθ. The enzyme maintains genomic stability and prevents tumorigenesis. It is a crucial enzyme in the mutagenic microhomology-mediated end joining (MMEJ) repair of DSBs, a pathway that plays critical role in genomic stability. Inhibiting PARP1, a key enzyme in MMEJ, prevents POLθ recruitment to sites of laser micro-irradiation. Since POLθ inhibition and PARPi have additive effects on HR-deficient cells, these data suggest that POLθ also functions outside the PARP-mediated MMEJ pathway and in pathways that are critical to the survival of HR-deficient cells. POLθ is a large protein that contains 3 domains and is structurally and functionally distinct from other polymerases. The N-term contains a helicase-like ATPase domain that can unwind several types of DNA structures, while the central domain binds RAD51, displaces RPA proteins from resected DSBs, and antagonizes HR repair in an ATP-hydrolysis dependent manner. Finally, the C-term contains a nuclease domain which trims DNA ends and a polymerase domain that fills in nucleotides during MMEJ. Both the ATPase and polymerase domains are required for MMEJ repair. POLθ has several functions that preserve genomic stability, and POLθ-mediated MMEJ repair can compensate for the loss of HR. It remains unclear which of the many functions of POLθ underlies the synthetic lethality with HR. Nevertheless, POLθ exhibits unique features of druggability, providing a strong rationale for developing POLθ inhibitors. We have recently performed a small-molecule screen for inhibitors of POLθ ATPase activity and identified the antibiotic novobiocin (NVB) as a specific and potent inhibitor of human POLθ. NVB binds to purified POLθ protein, prevents its recruitment to DNA damage, and inhibits MMEJ repair. Importantly, we have shown that NVB selectively kills HR-deficient (BRCA1- and BRCA2-deficient) cells over wild-type cells and potentiates the cytotoxic effect of PARPi in HR-deficient tumor cells in vitro and in vivo. Moreover, NVB kills HR-deficient, PARPi-resistant tumor cells. These results suggest that NVB can be used alone or in combination with PARPi for treating HR-deficient tumors, even after they have acquired PARPi resistance. Accordingly, clinical trials have now been initiated for the use of NVB in the management of these tumors. Citation Format: Alan d’Andrea. Pol-theta inhibitors in HRD tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED12-1.

Mechanisms of PARP Inhibitor Resistance.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) represent the first medicines based on the targeting of the DNA damage response (DDR). PARPi have become standard of care for first-line maintenance treatment in ovarian cancer and have also been approved in other cancer indications including breast, pancreatic and prostate. Despite their efficacy, resistance to PARPi has been reported clinically and represents a growing patient population with unmet clinical need. Here, we describe the various mechanisms of PARPi resistance that have been identified in pre-clinical models and in the clinic.

Fighting resistance: post-PARP inhibitor treatment strategies in ovarian cancer.

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a therapeutic milestone in the management of epithelial ovarian cancer. The concept of 'synthetic lethality' is exploited by PARPi in tumors with defects in DNA repair pathways, particularly homologous recombination deficiency. The use of PARPis has been increasing since its approval as maintenance therapy, particularly in the first-line setting. Therefore, resistance to PARPi is an emerging issue in clinical practice. It brings an urgent need to elucidate and identify the mechanisms of PARPi resistance. Ongoing studies address this challenge and investigate potential therapeutic strategies to prevent, overcome, or re-sensitize tumor cells to PARPi. This review aims to summarize the mechanisms of resistance to PARPi, discuss emerging strategies to treat patients post-PARPi progression, and discuss potential biomarkers of resistance.

Loss of Heterozygosity of BRCA1/2 as a Predictive Marker for Talazoparib Response.

Tumor cell lines are essential tools in understanding the molecular mechanisms underlying cancer biology and therapeutic responses. Poly (ADP-ribose) polymerase inhibitors (PARPi) kill tumor cells harboring pathogenic mutations of BRCA DNA repair-associated genes 1/2 (BRCA1/2) and are approved to treat ovarian and metastatic breast cancer. Loss of heterozygosity (LOH) of the wild-type BRCA1/2 locus is suspected to increase cellular response to PARPi. To better elucidate the molecular mechanisms underlying PARPi sensitivity and resistance, this study assessed the responses of various pathogenic BRCA1/2-mutant cell lines to the PARPi talazoparib. Mutant cell lines were extracted and cultured from four surgically resected, human breast cancer specimens with different pathogenic BRCA1/2, one normal breast specimen and one ovarian cancer specimen. Mutation analysis was performed on all cell lines using genomic DNA extraction and polymerase chain reaction. Following treatment with talazoparib, cell growth was assessed using tetrazolium salt and half-maximal inhibitory concentration values were determined. A partial correlation between different variants of pathogenic BRCA1/2 mutation and talazoparib susceptibility was found, with five of the cell lines exhibiting sensitivity to talazoparib. The most sensitive cell-line to talazoparib had LOH for BRCA1, while the breast cancer cell line harboring BRCA2 LOH was resistant to talazoparib. This study suggests that LOH does not necessarily correlate with PARPi efficacy. These results lay a foundation for future studies to utilize these novel cell lines to further elucidate the underlying molecular mechanisms of PARPi resistance and reveal new potential drug targets.

Mechanisms of PARP1 inhibitor resistance and their implications for cancer treatment.

The discovery of synthetic lethality as a result of the combined loss of PARP1 and BRCA has revolutionized the treatment of DNA repair-deficient cancers. With the development of PARP inhibitors, patients displaying germline or somatic mutations in BRCA1 or BRCA2 were presented with a novel therapeutic strategy. However, a large subset of patients do not respond to PARPinhibitors. Furthermore, many of those who do respond eventually acquire resistance. As such, combating de novo and acquired resistance to PARPinhibitors remains an obstacle in achieving durable responses in patients. In this review, we touch on some of the key mechanisms of PARPinhibitorresistance, including restoration of homologous recombination, replication fork stabilization and suppression of single-strandedDNA gap accumulation, as well as address novel approaches for overcoming PARPinhibitor resistance.

PARP inhibitor resistance in breast and gynecological cancer: Resistance mechanisms and combination therapy strategies.

Breast cancer and gynecological tumors seriously endanger women’s physical and mental health, fertility, and quality of life. Due to standardized surgical treatment, chemotherapy, and radiotherapy, the prognosis and overall survival of cancer patients have improved compared to earlier, but the management of advanced disease still faces great challenges. Recently, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have been clinically approved for breast and gynecological cancer patients, significantly improving their quality of life, especially of patients with BRCA1/2 mutations. However, drug resistance faced by PARPi therapy has hindered its clinical promotion. Therefore, developing new drug strategies to resensitize cancers affecting women to PARPi therapy is the direction of our future research. Currently, the effects of PARPi in combination with other drugs to overcome drug resistance are being studied. In this article, we review the mechanisms of PARPi resistance and summarize the current combination of clinical trials that can improve its resistance, with a view to identify the best clinical treatment to save the lives of patients.

BRCA reversion mutations mediated by microhomology-mediated end joining (MMEJ) as a mechanism of resistance to PARP inhibitors in ovarian and breast cancer.

5559 Background: PARP inhibitors exploit synthetic lethality in tumor cells with deficiency in homologous recombination repair (HRR). In line with this, most reported mechanisms of PARP inhibitor resistance restore HRR. Of multiple resistance mechanisms reported preclinically, reversion mutations in BRCA genes are the only confirmed mechanism of resistance to both platinum and PARP inhibitors in patients (pts) to date (Lin K et al. Cancer Discov 2019), with most studies focusing on ovarian cancer. MMEJ is an alternative DNA damage repair pathway, in which DNA polymerase θ (POLθ) has a key role; MMEJ has been suggested to play a role in BRCA reversion mutations (Tobalina L et al. Ann Oncol 2021). Methods: Targeted circulating tumor DNA (ctDNA) sequencing analyzed over 500 plasma samples collected at baseline and at progression to therapy in pts with ovarian or breast cancer and a mutation in BRCA1 and/or BRCA2 (BRCAm) who were treated with olaparib or chemotherapy in one of three Phase II/III clinical studies (LIGHT NCT02983799, SOLO3 NCT02282020, OlympiAD NCT02000622). Only pts with an original pathogenic BRCAm detected in ctDNA were evaluable. BRCA reversion mutations were identified using internal computational framework; DNA sequences surrounding BRCA reversion sites were analyzed for MMEJ signatures. Results: At baseline, in pooled data across treatment arms and across all available samples, BRCA reversion mutations were detected in 4/114 (3.5%) and 6/133 (4.5%) of breast and ovarian cancer pts, respectively, which may have developed on prior platinum therapy. At progression, BRCA reversion mutations were detected in 34/79 (43%) breast cancer pts and in 26/101 (26%) ovarian cancer pts who received olaparib, with at least 2/79 and 4/101 reversions already present at baseline, respectively. At progression, in the chemotherapy arm, BRCA reversion mutations were detected in 3/34 (9%) breast cancer pts and 1/29 (3%) ovarian cancer pts, with 2/34 and 0/29 reversions present at baseline, respectively. Reversion mutations varied in allelic frequency and were either present as single or multiple reversions, suggesting multiple events within the tumor were driving resistance. The location and type of reversion mutations reflected the functional importance of BRCA protein domains. A large proportion of BRCA reversion mutations (47/69 [68%] that were evaluable) were mediated by the MMEJ pathway based on the presence of MMEJ signatures around BRCA reversion sites. Conclusions: We detected BRCA reversion mutations in at least ̃40% of breast and ̃20% of ovarian cancer pts following treatment with olaparib. A large proportion of these reversion mutations are likely to have been mediated by MMEJ repair, suggesting that POLθ inhibitors in combination with platinum or PARP inhibitors might prevent or delay emergence of PARP inhibitor resistance. Clinical trial information: NCT02983799, NCT02282020, NCT02000622.

Exploring the mechanism of PARPi resistance in ovarian cancer by integrated analyze of pre- and post-progression samples in 5 patients.

e17582 Background: PARP inhibitors (PARPi) have been widely used in the treatment of homologous recombination repair-deficient tumors. Comprehensive investigations on the resistance mechanisms of PARPi and tumor microenvironment alterations after PARPi therapy are largely insufficient. Methods: 5 ovarian cancer patients (pts) treated with PARPi at West China Second University Hospital were enrolled in this study (Ethical Lot Number: 20200076). Clinicopathological information of the pts are shown in table below. Paired PARPi treatment-naive surgical samples (TNS) and the biopsy samples that progressed after PARPi maintenance therapy (PPS) were obtained. Multi-omic profiling was conducted by an NGS panel (Master Panel, AmoyDx) containing DNA exon regions of 563 genes and RNA expression of 1831 genes analyzing genetic mutations and dynamics of gene expression profile in these paired tumor samples. Results: Median duration of treatment with PARPi was 18 months (11-22). Comprehensive genomic profiling discovered MYC amplification (n = 3), ZFHX4 mutation (n = 2) and IDH1 mutation (n = 1) in PPS. One PPS harbored simultaneous amplification of PDCD1, FGF3, MYC, FGF19 and ZFHX4 mutation. In addition, BRCA1 intron11: c.4096+1G > T mutation was detected in one PPS carrying germline BRCA1 mutation (E1066*), which putatively resulted in restoration of BRCA1 function. At RNA level, single-sample enrichment analysis showed that B cells, T cells, and co-activation factors were significantly up-regulated in PPS. In addition, expression of CXCL13 and activation of CCL19/21-CCR7 axis were significantly increased, which indicated a PARPi-triggered “hot” immune-microenvironment. Moreover, immune-checkpoint TIGIT was significantly upregulated, paved the way for immunotherapy. Nevertheless, stromal remodeling, and tumor proliferation signatures were also up-regulated in PPS. In parallel, cancer-related pathways, PI3K/AKT, Epithelial-Mesenchymal transition, cell-cycle as well as G2M checkpoint were observed in the pathway enrichment analysis, which further verified the aforementioned findings. Conclusions: This small sample size study confirmed the complexity of the resistance mechanisms to PARPi, including BRCA reversion mutation, oncogene activation and bypass signaling activation. The activation of both positive and negative immune regulators in the tumor microenvironment shed light on future clinical management for patients with PARPi resistance.[Table: see text]

Targeting of RecQ Helicases as a Novel Therapeutic Strategy for Ovarian Cancer.

Simple SummaryOvarian cancer is the most lethal gynecologic malignancy and is characterized by genomic instability and DNA repair defects. PARP inhibitors (PARPi) changed the treatment paradigm of ovarian cancer but the development of resistance to PARPi is a pressing clinical challenge. In this review, we discuss how RecQ helicases can be targeted as a novel therapeutic strategy to prevent such treatment resistance. The combination of helicase inhibitor with a PARP inhibitor (PARPi) or ATR inhibitor may overcome PARPi resistance in ovarian cancer.RecQ helicases are essential for DNA replication, recombination, DNA damage repair, and other nucleic acid metabolic pathways required for normal cell growth, survival, and genome stability. More recently, RecQ helicases have been shown to be important for replication fork stabilization, one of the major mechanisms of PARP inhibitor resistance. Cancer cells often have upregulated helicases and depend on these enzymes to repair rapid growth-promoted DNA lesions. Several studies are now evaluating the use of RecQ helicases as potential biomarkers of breast and gynecologic cancers. Furthermore, RecQ helicases have attracted interest as possible targets for cancer treatment. In this review, we discuss the characteristics of RecQ helicases and their interacting partners that may be utilized for effective treatment strategies (as cancers depend on helicases for survival). We also discuss how targeting helicase in combination with DNA repair inhibitors (i.e., PARP and ATR inhibitors) can be used as novel approaches for cancer treatment to increase sensitivity to current treatment to prevent rise of treatment resistance.

PARP Inhibition Activates STAT3 in Both Tumor and Immune Cells Underlying Therapy Resistance and Immunosuppression In Ovarian Cancer.

Despite the promising activity of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) in many cancer types with defects in the DNA damage response the majority of the treated patients acquire PARPi resistance and succumb to their diseases. Consequently, there is an urgent need to identify the mechanisms of PARPi resistance. Here, we show that PARPi treatment promotes STAT3 activation in ovarian cancer cells, tumor-associated immune cells and fibroblasts, resulting in PARPi resistance and immunosuppression. Comparison of ovarian cancer patient-matched tumor biopsies before and after PARPi therapy revealed that STAT3 activity was significantly higher in tumor cells and tumor-associated immune cells and fibroblasts post PARPi treatment. Moreover, one-time PARPi treatment activated STAT3 both in tumor cells as well as diverse immune subsets and fibroblasts. PARPi-treated immune cells exhibited decreased expression of immunostimulatory interferon (IFN)-γ and Granzyme B while increasing immunosuppressive cytokine IL-10. Finally, we demonstrate that the acquisition of PARPi resistance in ovarian cancer cells was accompanied by increased STAT3 activity. Ablating STAT3 inhibited PARPi-resistant ovarian tumor cell growth and/or restored PARPi sensitivity. Therefore, our study has identified a critical mechanism intrinsic to PARPi that promotes resistance to PARPi and induces immunosuppression during PARPi treatment by activating STAT3 in tumor cells and tumor-associated immune cells/fibroblasts.