HR-deficient Tumors Research Articles

Performance of a RAD51-based functional HRD test on paraffin-embedded breast cancer tissue

PurposeBRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR deficiency (HRD) extends beyond BRCA-associated BC, highlighting the need for a sensitive method to enrich for HRD tumors in an alternative way. A promising approach is the use of functional HRD tests which evaluate the HR capability of tumor cells by measuring RAD51 protein accumulation at DNA damage sites. This study aims to evaluate the performance of a functional RAD51-based HRD test for the identification of HRD BC.MethodsThe functional HR status of 63 diagnostic formalin-fixed paraffin-embedded (FFPE) BC samples was determined by applying the RAD51-FFPE test. Samples were screened for the presence of (epi)genetic defects in HR and matching tumor samples were analyzed with the RECAP test, which requires ex vivo irradiated fresh tumor tissue on the premise that the HRD status as determined by the RECAP test faithfully represented the functional HR status.ResultsThe RAD51-FFPE test identified 23 (37%) of the tumors as HRD, including three tumors with pathogenic variants in BRCA1/2. The RAD51-FFPE test showed a sensitivity of 88% and a specificity of 76% in determining the HR-class as defined by the RECAP test.ConclusionGiven its high sensitivity and compatibility with FFPE samples, the RAD51-FFPE test holds great potential to enrich for HRD tumors, including those associated with BRCA-deficiency. This potential extends to situations where DNA-based testing may be challenging or not easily accessible in routine clinical practice. This is particularly important considering the potential implications for treatment decisions and patient stratification.

Ovarian high-grade serous carcinoma with transitional-like (SET) morphology: a homologous recombination-deficient tumor

Thirteen years ago, we pointed out that ovarian transitional cell carcinomas (TCCs) and conventional high-grade serous carcinomas (HGSCs) had similar genetic alterations and clinical behavior. Consequently, ovarian TCC is now classified as a morphologic variant of HGSC. Defective homologous recombination, resulting from genetic or epigenetic inactivation of DNA damage repair genes, such as BRCA1/2, occurs in approximately 50% of the HGSCs. Although BRCA mutations have been associated with HGSCs with solid, pseudoendometrioid or transitional (SET) features, little is known about the role of non-BRCA homologous recombinationrepair (HRR) genes and the HRR status in these tumors. Using two commercially available assays (Myriad Genetics MyChoice CDx Plus test and SOPHiA Dx Homologous Recombination Deficiency Solution), we study mutations of BRCA1/2 and non-BRCA HRR genes (ATM, BARD1, BRIP1, CDK12, CHEK1/2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), and the HRR status in 19 HGSCs with SET features and 20 HGSCs with classic morphology. We also studied, as control cases, 5 endometrioid carcinomas, 1 clear cell carcinoma, 2 low-grade serous carcinomas, and 1 malignant Brenner tumor. Seven HGSCs with SET features (7/19; 37%) showed BRCA mutations (4 BRCA1, 2 BRCA2, and 1 BRCA1/2). Mutations in non-BRCA HRR genes were found in ATM (1/15; 7%), BARD1 (1/15; 7%), and BRIP1 (1/19; 5%). MostHGSCs with SET features (17/19; 90%) were considered to be homologous recombination-deficient tumors. Three HGSCs with classic morphology (3/20; 15%) showed BRCA2 mutations. Mutations in non-BRCA HRR genes were found in CDK12 (2/14; 14%), FANCL (1/14; 7%), RAD51B (1/14; 7%), and RAD54L (1/14; 7%). Eleven HGSCs with classical morphology (11/20; 55%) were considered to be homologous recombination deficient. In contrast, all ovarian carcinoma control cases (5 endometrioid carcinomas, 1 clear cell carcinoma, 2 low-grade serous carcinomas, and 1 malignant Brenner tumor) were homologous recombination proficient and did not have BRCA mutations. Our results show that the majority of HGSCs with SET features are homologous recombination-deficient tumors independently of the BRCA status and highlight the importance of the HRR tumor testing, especially in BRCA wild-type tumors. Recognition of transitional cell variant of HGSCs may help to identify patients most likely to benefit from PARP inhibitors.

ShallowHRD status acts as an effective prognostic predictor in ovarian cancer patients treated by poly (ADP-ribose) polymerase inhibitors (PARPis).

Homologous recombination deficiency (HRD) plays a crucial role in ovarian cancer patients who are treated with Poly (ADP-ribose) polymerase inhibitors (PARPis). It could be defined as a prognosis biomarker. However, many high throughput sequencing methods for evaluating HRD, including HRDetect (WGS 10X), SigMA (WGS 40X or panel 1000X), and scarHRD (WGS 30X), are technically complex, time and data-storage consuming, and costly. Herein, we aimed to develop a low-cost method by low sequencing coverage to identify HRD status for precision medication. We utilized ShallowHRD, a software tool to evaluate tumor HRD based on whole genome sequencing (WGS) at low coverage (1X), and established a novel scoring system, ShallowHRD score system. Compared with negative ShallowHRD status (ShallowHRD score < 15 or BRCAwild), positive ShallowHRD status (ShallowHRD score ≥ 15 or BRCAmut) presented favorable survival after being treated with PARPis. The ShallowHRD status is a good biomarker for predicting prognosis, which could help guide the clinical application of PARPis in ovarian cancer patients by a cost-effective, time and data-storage saving method.

Identification of immunotherapy biomarkers for improving the clinical outcome of homologous recombination deficiency patients with lung adenocarcinoma.

Homologous recombination deficiency (HRD) is a common molecular signature of genomic instability and has been shown to be a biomarker for targeted therapies. However, there is a lack of studies on the role of HRD changes in lung adenocarcinoma (LUAD) transcriptomics. HRD scores were determined using single nucleotide polymorphism (SNP) array data from LUAD patients from The Cancer Genome Atlas (TCGA) database. Transcriptional data from patients with different scores were analyzed to identify biomarkers associated with HRD. Candidate biomarkers were validated using Gene Expression Omnibus (GEO)-sourced datasets and an immunotherapy cohort. According to the bulk transcriptome and clinical characteristics of 912 LUAD patients and Single-cell RNA-seq of 9 LUAD patients from TCGA and GEO databases, we observed increased MS4A6A expression in HRD tumors; high MS4A6A expression predicted improved survival outcomes. Furthermore, a comprehensive analysis of the tumor immune microenvironment (TIME) revealed a positive correlation between MS4A6A expression and neoantigen loading and immune cell infiltration. Additionally, the immunotherapy cohort confirmed the possibility of using MS4A6A as a biomarker. Collectively, we suggest that MS4A6A is associated with HRD and provide a new perspective toward identifying promising biomarkers for immunotherapy.

FIRRM/C1orf112 mediates resolution of homologous recombination intermediates in response to DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) pose a major obstacle for DNA replication and transcription if left unrepaired. The cellular response to ICLs requires the coordination of various DNA repair mechanisms. Homologous recombination (HR) intermediates generated in response to ICLs, require efficient and timely conversion by structure-selective endonucleases. Our knowledge on the precise coordination of this process remains incomplete. Here, we designed complementary genetic screens to map the machinery involved in the response to ICLs and identified FIRRM/C1orf112 as an indispensable factor in maintaining genome stability. FIRRM deficiency leads to hypersensitivity to ICL-inducing compounds, accumulation of DNA damage during S-G2 phase of the cell cycle, and chromosomal aberrations, and elicits a unique mutational signature previously observed in HR-deficient tumors. In addition, FIRRM is recruited to ICLs, controls MUS81 chromatin loading, and thereby affects resolution of HR intermediates. FIRRM deficiency in mice causes early embryonic lethality and accelerates tumor formation. Thus, FIRRM plays a critical role in the response to ICLs encountered during DNA replication.

Homologous recombination deficiency (HRD) status and response to platinum-based chemotherapy and PARP inhibitors in Chinese patients with ovarian cancer.

e17533 Background: The association between platinum-based chemotherapy and PARPis response and HRD status in Chinese epithelial ovarian cancer patients has not been fully elucidated. Methods: We assessed homologous recombination deficiency (HRD) score using genomic scar analysis (GSA) algorithm of Beijing Genomics Institute (BGI) assay. The definitive HRD status were determined by combining HRD score tested with the BGI assay and homologous recombination repair (HRR) gene mutation status. We then tested the association of HRD with response to platinum-based chemotherapy and PARP inhibitors in Chinese patients with ovarian cancer. Results: In total, 209 patients with definitive HRD status were included: 66.0% (138 of 209) for HRD and 34.0% (71 of 209) for non-HRD. Patients with HRD tumors were more likely to have a larger tumor (73.9% vs 59.2%, P = 0.029), and less likely to receive neoadjuvant chemotherapy (48.6% vs 67.6%, P = 0.009). No differences were found between patients with HRD and non-HRD tumors in age at diagnosis, histology, stage, postoperative residual disease status. In the entire cohort, patients with HRD tumors were more likely to be platinum sensitive with a platinum-free interval (PFI) of &gt; 6 months than those with non-HRD (79.6% vs 65.7%, P=0.030). Patients with HRD had a significantly better PFS and OS than patients with non-HRD in the entire study population (PFS: unadjusted HR = 0.54, 95% CI: 0.38–0.77, P = 0.001; and OS: unadjusted HR = 0.44, 95% CI: 0.26–0.75, P = 0.002). Multivariate analysis showed that patients with HRD was an independent predictor of increased PFS and OS (PFS: HR = 0.56, 95% CI: 0.39-0.82, P = 0.002; OS: HR = 0.50, 95% CI: 0.29-0.86, P = 0.012). In patients who received PARPis as first-line maintenance (n=55), the median duration of PARPis was 23.3 months (1.2-40.0 months) and 11.0 months (1.4-39.1 months) in HRD and non-HRD subgroup, respectively. Patients with HRD had a significantly better PFS than non-HRD patients (median PFS: NR vs 22.5 months, unadjusted HR = 0.39, 95% CI: 0.16-0.97, P = 0.035). Multivariate analysis showed that patients with HRD was an independent predictor of increased PFS (HR = 0.22, 95% CI: 0.07-0.68, P = 0.008). Conclusions: Patients with HRD tumors are more likely to respond to platinum-based chemotherapy and PARPis when compared with those who are non-HRD. HRD tumors associate with a better survival in Chinese ovarian cancer patient.

Effect of homologous recombination deficient (HRD) breast cancers on a distinct immune marker phenotype by comprehensive genomic and immune profiling (CGIP).

1029 Background: Preclinical evidence suggests that breast cancer with features of genomic instability may upregulate the host antitumor immune response by producing neoantigens through DNA damage and increasing interferon production through the stimulator of interferon genes (STING) pathway. In this study, we evaluated the association between features of genomic instability and immune response in a real-world breast cancer population. Methods: We analyzed retrospective comprehensive genomic and immune profiling (CGIP) results from 529 breast tumors tested in the real-world clinical setting. We defined the HRD phenotype as tumor with any single nucleotide variants (SNV), indels, copy number variations (CNV) or fusions in the following genes: ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CHEK1/2, FANCA, MRE11A, NBN, PALB2, RAD50 and RAD51. Otherwise, they were considered HR-proficient (HRP). PD-L1 expression (CPS positive ≥10) and TMB (high ≥10 Mut/Mb) were determined using IHC and DNA sequencing, respectively. mRNA expression signatures of tumor inflammation (TIGS, strong/moderate/weak), cell proliferation (CP, high/moderate/poor) and cancer testis antigen burden (CTAB, high/low) were determined by RNA-sequencing from a 395-gene panel. We used over-representation and proportion analysis using chi-squared test to determine association of HRD to immune correlates. Results: Among 529 cases, the median patient age was 63.2 years (25.5-93.5). The majority of patients were female (519, 98%), and the most common tumor histology was invasive ductal carcinoma (287, 54%). A total of 405 (77%) and 124 (23%) of patients had HRD and HRP phenotypes, respectively. A greater proportion of HRD tumors (16%) had higher TMB compared to HRP (5.6%, p=0.003). HRP tumors showed a relatively low CP index, whereas HRD tumors were associated with a moderate CP index score (p = 0.007). HRD phenotype was associated with a significantly higher proportion of weakly inflamed tumors, as represented by lower TIGS scores (p=0.007). No significant difference in PD-L1 or CTAB was found between HRD and HRP phenotypes. Conclusions: Breast tumors with mutations in the HR genes demonstrated greater TMB and moderate cellular proliferation index of both tumor and immune cells, which suggest susceptibility to immune checkpoint inhibitors. Interestingly, this cohort lacked elevated markers of immune infiltration (TIGS), indicating a mechanism of potential tumor immune evasion. These results suggest that CGIP may allow for more informed treatment decisions in HRD breast cancers. Furthermore, assessment of HR status and immunotherapy susceptibility may support clinical trial selections for therapies targeting the complex interplay of genomic and immune components of breast cancer (e.g., a combination of PARP inhibitor and immunotherapy).

Real-world Outcomes Associated With Poly(ADP-ribose) Polymerase Inhibitor Monotherapy Maintenance in Patients With Primary Advanced Ovarian Cancer.

This study used real-world population data to assess the trends of first-line (1L) poly(ADP-ribose) polymerase inhibitor (PARPi) maintenance treatment uptake and outcomes in patients with primary advanced ovarian cancer (AOC). Patients diagnosed with AOC between January 1, 2017, and June 30, 2021, who completed 1L chemotherapy were selected from a real-world database. Descriptive analyses were performed to evaluate patient demographics, clinicopathological characteristics, and 1L treatment patterns. Time to next treatment or death was used as a proxy for real-world progression-free survival (rwPFS). Kaplan-Meier methods and Cox models were used for statistical analyses. Of 705 patients who completed 1L chemotherapy, 166 received PARPi monotherapy and 539 underwent active surveillance (AS). Median follow-up was 10.9 months for PARPi monotherapy and 20.6 months for AS. PARPi monotherapy use increased from 6% in 2017 to 53% in 2021. Overall, patients receiving PARPi monotherapy had longer rwPFS than those who underwent AS (not reached vs 9.53mo) respectively. rwPFS was also longer in patients who received PARPi monotherapy compared with AS in patients with BRCA- mutated disease (not reached vs 11.4mo), BRCA- wild-type disease (13.5 vs 9.1mo), homologous recombination-deficient tumors (not reached vs 10.2mo), and homologous recombination-proficient or unknown status tumors (13.5 vs 9.3mo). Our real-world analysis suggested that 47% of patients with primary AOC did not receive PARPi maintenance in the year 2021. PARPi use was associated with significantly improved outcomes compared with AS.

Abstract 6398: Targeting ENPP1 to boost anti-tumor immunity during PARP inhibition

Abstract Homologous recombination (HR) deficiency confers sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPi), which have been approved for use in ovarian and triple negative breast cancers (TNBC) carrying loss-of-function mutations in HR pathway genes, most commonly BRCA1/2. Preclinical studies in BRCA1-deficient tumor models have demonstrated that the immune compartment is required for PARPi efficacy through the ability of PARPi to cause micronuclei formation and activate cGAS in tumors cells. The production of the 2’3’-cGAMP secondary intermediate stimulates STING in neighboring tumor-infiltrating immune cells to promote an anti-tumor response. Despite this evidence of immune stimulation, PARPi does not extend overall survival in TNBC patients, suggesting that immune suppression remains a significant barrier for efficacy. Here we sought to evaluate the role of ectonucleotide pyrophosphatase/phosphodiesterase 1(ENPP1), a type II transmembrane glycoprotein that hydrolyzes extracellular 2’3’-cGAMP. We generated Brca1/Pten/Trp53-deficient TNBC cell lines from a genetically engineered mouse model and found that these expressed higher levels of ENPP1 than Brca1-proficient TNBC and non-TNBC cell lines. Knocking out Enpp1 via CRISPR/Cas9 increased 2’3’-cGAMP production during Niraparib treatment but did not affect cellular proliferation in vitro. Critically, knocking out Enpp1 significantly reduced the growth of orthotopic Brca1-deficient tumors, with no change in growth observed for orthotopic E0771 or PyMT models, suggesting an important role for ENPP1 in HR-deficient tumors. Therapeutic dosing of Niraparib further reduced the growth of Enpp1 knockout tumors in a manner that was dependent upon host STING and correlated with higher CD8+ T cell infiltration and effector function. These findings demonstrate that ENPP1 suppresses anti-tumor immunity in Brca1-deficient tumors, suggesting that blocking the enzymatic activity of ENPP1 may provide therapeutic benefits during PARPi treatment for TNBC patients. Citation Format: Jie Li, Charlotte Mason, Kay Hanggi, Vincent Luca, Brian Ruffell. Targeting ENPP1 to boost anti-tumor immunity during PARP inhibition. [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 6398.

Abstract 6200: PARP1 hyperactivation by the decoy oligodeoxynucleotide OX425 mediates DNA repair abrogation and unleashes the anti-tumor immune response

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPis) lead to synthetic lethality when used in cancers with homologous recombination deficiency (HRD). However, the development of resistance to PARPis is a recurrent problem thus limits the duration of response and hence the clinical utility of these agents. Here, we describe the antineoplastic and immunomodulatory effects of OX425, a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy, resulting in constitutive PARP1 hyperactivation and consequent exhaustion of the DNA damage response. Methods: OX425-induced PARP trapping, hyperactivation and cell cytotoxicity were examined in vitro in HRD and homologous recombination proficient (HRP) human cancer cells, as well as in non-transformed cell lines. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. OX425 effects on the innate and adaptive immune responses were assessed by following STING activation and T-cell mediated anti-tumor cytotoxicity. RNAseq analysis in HRP/HRD tumor cells treated with OX425 or PARP inhibitors was employed to uncover the molecular mechanisms underlying OX425 effects. The anticancer efficacy of OX425 was assessed in vivo in different HRD and HRP tumor models. OX425-induced PARP activation and tumor infiltration by immune cells were analyzed by flow cytometry. Results: At odds with conventional PARP inhibitors, OX425 bound to and hyperactivated PARP1 with high affinity in a dose-dependent manner, resulting in elevated cytotoxicity to multiple cancer cells (breast, ovarian, prostate, colon, hematological, endometrial cancers) irrespective of HR status. Interestingly, long-term treatment with OX425 did not show any mutagenicity compared to PARPi. The activity of OX425 was specific to tumor cells, as no significant effect on cell viability was observed for normal cells, at odds with PARP inhibitors. In line with in vitro results, OX425 mediated considerable anticancer effects in vivo. Moreover, OX425 triggered activation of the STING pathway and CCL5 secretion in the EMT6 mouse mammary carcinoma model. The anticancer effect of OX425 was coupled with tumor-targeting T cell responses. In MPA/DMBA-driven mammary tumors, OX425 mediated considerable anticancer effects in monotherapy and synergistic effects in combination with PD1 inhibition. Moreover, OX425 treatment significantly delayed acquired resistance to olaparib in BRCA1 mutated MDA-MB-436 cell-derived xenografts. Conclusions: Our results provide preclinical rationale for using OX425 to trigger DNA damage exhaustion and STING activation in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD or HRP tumors Citation Format: Vlada Zakharova, Claudia Galassi, Chloé Doizelet, Vincent Hayes, Lorenzo Galluzzi, Wael Jdey. PARP1 hyperactivation by the decoy oligodeoxynucleotide OX425 mediates DNA repair abrogation and unleashes the anti-tumor immune response [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 6200.

Abstract 512: Identification of SS008871, a novel Polθ inhibitor that effectively inhibits tumors with homologous recombination deficiency in vitro and in vivo

Abstract The repair of DNA double strand break (DSB) is crucial for genome stability and cell survival. There are three main DSB repair pathways: homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). HR is a high-fidelity, error-free DSB repair pathway, and the dysfunction of HR confers cell genome instability and leads to tumorigenesis. HR deficiency is especially prevalent in gynecologic tumors, sharing about 10% population of all the gynecologic tumor patients. In situation of HR deficiency, MMEJ, in which DNA polymerase theta (Polθ) plays an essential role, 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. Hence, Polθ emerges as a potential DNA damage repair target for the treatment of HR deficient tumors. Here we report a novel small molecular Polθ inhibitor, SS008871, which inhibits Polθ activity with an IC50 of 22 nM, and strongly inhibits cellular MMEJ pathway with an IC50 of single-digital nanomolar level. SS008871 strongly inhibits proliferation of HR deficient BRCA2-/- DLD-1 cells, and shows a &amp;gt;125× selectivity folds over DLD-1 parent cells as well as non-malignant cells. Besides, SS008871 elicits synergetic anti-proliferation activities in combination of a PARP inhibitor, olaparib on BRCA2-/- DLD-1 and MDA-MB-436 cells. In the BRCA2-/- DLD-1 xenograft model, SS008871 shows tumor growth inhibition as a single agent, and the combination of SS008871 and olaparib further results in tumor regression. Accordingly, the level of γH2AX, a common DSB marker, correlates well to the anti-tumor efficacy. In a human hematopoietic stem cells based in vitro hematotoxicity assay, SS008871 shows no significant inhibition on lineage-specific (myeloid, erythroid and megakaryocytic) cell differentiation and survival, suggesting the low hematotoxicity risk. In comparison, olaparib significantly attenuated hematopoietic stem cells on both differentiation and survival in the parallel assay, which is consistent with the hematological toxicity observed in human. Furthermore, there is no clinical abnormalities observed after a high dose treatment of SS008871 in mice, demonstrating that SS008871 is well-tolerated. Taken together, SS008871 is proved to be an encouraging Polθ inhibitor with good safety. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Feng Tang, Zhen Li, Wenqing Yang, Liting Xue, L Chen, Renhong Tang. Identification of SS008871, a novel Polθ inhibitor that effectively inhibits tumors with homologous recombination deficiency in vitro and in vivo [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 512.

Abstract 6112: Automated scoring to assess RAD51-mediated homologous recombination in patient-derived tumor organoids of ovarian cancers

Abstract Extensive trials of PARP inhibitors (PARPi) have shown that they improve progression-free survival in patients with various cancers, including ovarian cancers. This improvement is marked in patients carrying BRCA mutations, and in Homologous Recombination Deficiency (HRD) subgroups. To date, identifying patients eligible for PARPi has been a challenge for scientific and clinical teams using next-generation sequencing (NGS) analysis and the persistence of genomic scars in tumors after restoration of proficient HR or epigenetic changes is a limitation of NGS. Functional assays could be used to improve the profiling of HR status and faithfully identify HRD tumors. The Repair Capacity Test assesses the formation of RAD51 foci in proliferating cells after irradiation and can be used on fresh primary cancer tissues irradiated ex vivo as well as on Patient-Derived Tumor Organoids (PDTO). However, RAD51 foci scoring is often performed manually without any possibility for the standardization of techniques. By contrast, recent progress in whole slide imaging referring to scan a complete microscope slide and creating a single high-resolution digital file, could represent an opportunity for automatizing the evaluation of HR. The purpose of this translational study was to develop an automated tool for scoring RAD51-mediated homologous recombination, to use this tool on ovarian PDTO and to compare the result to the sensitivity to olaparib (PARPi), determined by direct exposure of PDTO to the drug. We show that immunofluorescence of RAD51 foci can be automatically detected and quantified in Cyclin A2 positive nuclei in all the cells of each PDTO slice thus offering a new opportunity for the routine management and standardization of HR assessment in PDTO that goes beyond the widely used manual estimation. Finally, this automated scoring will be optimized to directly assess HR on tumor slices to make the Repair Capacity Test a highly relevant functional precision oncology tool to identify patients who will benefit from PARPi. Citation Format: Lucie Thorel, Pierre-Marie Morice, Nicolas Elie, Louis-Bastien Weiswald, Romane Florent, Florence Giffard, Margaux Jacobs, Agathe Ricou, Raphaël Leman, Guillaume Babin, Jean-François Lebrun, Sandrine Martin, Mélanie Briand, Benoit Goudergues, Bernard Lambert, Cécile Blanc-Fournier, Dominique Vaur, Benoit Plancoulaine, Laurent Poulain. Automated scoring to assess RAD51-mediated homologous recombination in patient-derived tumor organoids of ovarian cancers [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 6112.

Abstract 5610: [18F]FluorThanatrace ([18F]FTT) PET Imaging of PARP-inhibitor drug-target engagement as a biomarker of response in ovarian cancer

Abstract Purpose: Poly(ADP-ribose) polymerase enzyme inhibitors (PARPi) have become the standard-of-care treatment for homologous recombination deficient (HRD) high-grade serous ovarian cancer (HGSOC). However, not all HRD tumors respond to PARPi and biomarkers to predict response are needed. [18F]FluorThanatrace (FTT) is a PARPi-analog PET radiotracer that non-invasively measures PARP-1 expression. Herein, we evaluate the ability of FTT uptake to serve as a biomarker to predict response to PARPi in patient-derived xenograft (PDX) models and patients with HGSOC. Patients and Methods: In PDX models, FTT-PET was performed before and after PARPi (olaparib), ataxia-telangiectasia inhibitor (ATRi), or both. Changes in FTT were correlated with tumor size changes. Patients with HRD and HGSOC that were enrolled in CAPRI (PARPi+ATRi), LIGHT (PARPi only), or off-trial (PARPi only) were selected for this single-center, prospective, cohort, IRB-approved study. FTT-PET/CT imagining was obtained from the skull base to the proximal thighs on an Ingenuity TF scanner (Philips Healthcare) 60-90 minutes after intravenous infusion of 8-12 mCi FTT. Subjects were imaged with FTT-PET at baseline and after ~1 week of PARPi monotherapy treatment. Target lesions (primary tumor and/or metastases) were identified at the time of the baseline imaging on correlative anatomic imaging using RECIST 1.1 and maximum standardized uptake value (SUVmax) data, normalized by body weight, was collected. Changes in FTT-PET uptake were compared to changes in tumor size, CA-125, and progression free survival (PFS). Results: A decrease in FTT tumor uptake after 1 week of PARPi treatment correlated with response to PARPi+ATRi treatment in PARPi-resistant PDX models (r=0.81-0.83, P=0.1-0.22). In HGSOC patients (n=13), percent differences in FTT-PET after ~7 days of PARPi compared to baseline correlated with the first RECIST response (r=0.60, P=0.034), best RECIST response (r=0.75, P=0.01), best CA-125 response (r=0.73, P=0.033), and PFS (r=0.67, P=0.027). All patients with &amp;gt;50% reduction in FTT uptake had &amp;gt;6-month PFS and &amp;gt;50% reduction in CA-125 (P=0.004 and P=0.016, respectively). Utilizing only baseline FTT uptake correlated to best RECIST response (r=-0.65, P=0.035) but did not predict response when corelated with other measures. Importantly, a decrease in FTT uptake does not appear to be associated with a reduction in tumor burden or apoptosis in response to drug cytotoxic activity at this early timepoint, indicating specificity for drug-target engagement. Conclusions: The decline in FTT uptake shortly after PARPi initiation compared to baseline provides an in vivo measure of drug-target engagement and shows promise as an early biomarker to guide PARPi therapy. FTT-PET has both pre-clinical and clinical applications warranting further study, including guiding PARPi combination therapy. Citation Format: Sarah B. Gitto, Austin R. Pantel, Mehran Makvandi, Hyoung Kim, Sergey Medvedev, Joanna K. Weeks, Drew A. Torigian, Chia-Ju Hsieh, Benjamin Ferman, Nawar A. Latif, Janos L. Tanyi, Lainie P. Martin, Shannon M. Lanzo, Fang Liu, Quy Cao, Gordon B. Mills, Robert K. Doot, David A. Mankoff, Robert H. Mach, Lilie L. Lin, Fiona Simpkins. [18F]FluorThanatrace ([18F]FTT) PET Imaging of PARP-inhibitor drug-target engagement as a biomarker of response 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 5610.

Abstract 6190: Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers

Abstract Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [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 6190.

Homologous recombination deficiency signatures in gastrointestinal and thoracic cancers correlate with platinum therapy duration

There is emerging evidence about the predictive role of homologous recombination deficiency (HRD), but this is less defined in gastrointestinal (GI) and thoracic malignancies. We reviewed whole genome (WGS) and transcriptomic (RNA-Seq) data from advanced GI and thoracic cancers in the Personalized OncoGenomics trial (NCT02155621) to evaluate HRD scores and single base substitution (SBS)3, which is associated with BRCA1/2 mutations and potentially predictive of defective HRD. HRD scores were calculated by sum of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions scores. Regression analyses examined the association between HRD and time to progression on platinum (TTPp). We included 223 patients with GI (n = 154) or thoracic (n = 69) malignancies. TTPp was associated with SBS3 (p < 0.01) but not HRD score in patients with GI malignancies, whereas neither was associated with TTPp in thoracic malignancies. Tumors with gBRCA1/2 mutations and a somatic second alteration exhibited high SBS3 and HRD scores, but these signatures were also present in several tumors with germline but no somatic second alterations, suggesting silencing of the wild-type allele or BRCA1/2 haploinsufficiency. Biallelic inactivation of an HR gene, including loss of XRCC2 and BARD1, was identified in BRCA1/2 wild-type HRD tumors and these patients had prolonged response to platinum. Thoracic cases with high HRD score were associated with high RECQL5 expression (p ≤ 0.025), indicating another potential mechanism of HRD. SBS3 was more strongly associated with TTPp in patients with GI malignancies and may be complementary to using HRD and BRCA status in identifying patients who benefit from platinum therapy.

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.

Influence of Usnic Acid Derivative (Tyrosyl-DNA Phosphodiesterase 1 Inhibitor) on Transplanted Tumors in vivo as a Monotherapy and in Combination with Olaparib

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that removes various adducts from the 3'‑end of DNA. Such damage is formed, for example, under the action of enzymes that introduce single-strand breaks in DNA during catalysis (for example, topoisomerase 1), as well as a number of anticancer drugs with different mechanisms of action. Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme that catalyzes the post-translational modification of various targets (PARylation), and with its help controls many processes in the cell, including DNA repair. The target of PARP1 is also Tdp1, whose PARylation attracts Tdp1 to the site of DNA damage. Olaparib is a PARP1 inhibitor used in clinical practice to treat homologous recombination deficient tumors. The main mechanism of action of olaparib is to obstruction of PARylation and thus DNA repair. In this study, we used the Tdp1 inhibitor OL7-43 in combination with olaparib to increase the antitumor effect of the latter. Despite the increase in cytotoxicity of olaparib in the presence of OL7-43 in vitro, we did not find a sensitizing effect of this compound in the Lewis and Krebs-2 carcinoma models, but it showed its own antitumor and antimetastatic effects.

Functional Homologous Recombination Assay on FFPE Specimens of Advanced High-Grade Serous Ovarian Cancer Predicts Clinical Outcomes.

Deficiency in homologous recombination (HR) repair of DNA damage is characteristic of many high-grade serous ovarian cancers (HGSC). It is imperative to identify patients with homologous recombination-deficient (HRD) tumors as they are most likely to benefit from platinum-based chemotherapy and PARP inhibitors (PARPi). Existing methods measure historical, not necessarily current HRD and/or require high tumor cell content, which is not achievable for many patients. We set out to develop a clinically feasible assay for identifying functionally HRD tumors that can predict clinical outcomes. We quantified RAD51, a key HR protein, in immunostained formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained from chemotherapy-naïve and neoadjuvant chemotherapy (NACT)-treated HGSC patients. We defined cutoffs for functional HRD separately for these sample types, classified the patients accordingly as HRD or HR-proficient, and analyzed correlations with clinical outcomes. From the same specimens, genomics-based HRD estimates (HR gene mutations, genomic signatures, and genomic scars) were also determined, and compared with functional HR (fHR) status. fHR status significantly predicted several clinical outcomes, including progression-free survival (PFS) and overall survival (OS), when determined from chemo-naïve (PFS, P < 0.0001; OS, P < 0.0001) as well as NACT-treated (PFS, P < 0.0001; OS, P = 0.0033) tumor specimens. The fHR test also identified as HRD those PARPi-at-recurrence-treated patients with longer OS (P = 0.0188). We developed an fHR assay performed on routine FFPE specimens, obtained from either chemo-naïve or NACT-treated HGSC patients, that can significantly predict real-world platinum-based chemotherapy and PARPi response. See related commentary by Garg and Oza, p. 2957.

Circulating tumor DNA and homologous recombination deficiency in bone-predominant mCRPC prior to radium-223 therapy.

203 Background: Radium-223 (Ra-223) is a bone-seeking alpha emitter that induces double-stranded DNA breaks, and the homologous recombination (HR) pathway is critical for repairing these breaks. While prior studies suggested that metastatic castrate-resistant prostate cancers (mCRPC) patients (pts) with HR-deficient (HRD+) tumors may be more likely to benefit from Ra-223, obtaining tissue for next generation sequencing to identify HRD+ is challenging in pts with bone-predominant disease. We hypothesized that circulating tumor DNA (ctDNA) would allow for broader identification of HRD+ to assess association with clinical outcomes in a real-world cohort. Methods: We identified 135 mCRPC pts treated with Ra-223 at our institution between 2013 and 2021. Pts who initiated another anti-tumor therapy within 60 days of Ra-223 treatment were excluded; pts continuing hormonal agents initiated &gt;60 days prior were included. ctDNA isolated from pre-treatment plasma underwent ultra-low-pass whole genome sequencing to estimate tumor fraction (TF). Additionally, targeted panel sequencing using an institutional prostate cancer-specific panel of 319 genes with duplex sequencing (utilizing unique molecular identifiers) for error suppression was used to identify germline or somatic deleterious alterations in HR pathway genes. The primary outcome was association between HRD status and completion of fewer than 6 cycles (as a proxy for early clinical progression), assessed using logistic regression. Results: The median age was 61 (IQR, 56-67) years, median pretreatment prostate-specific antigen (PSA) level was 26.2 (IQR, 8.1-84.1) ng/mL, and median TF was 4% (IQR, 3-6%). 97% of pts (n=131) previously received a novel antiandrogen, and 63% (n=85) received prior taxane. 17% (n=23) were HRD+, and 59% (n=80) completed 6 cycles of Ra-223. On multivariable analysis, HRD+ was associated with decreased likelihood of completing 6 cycles compared to HRD- (adjusted odds ratio [AOR] 0.16, 95% confidence interval [CI] 0.05-0.48, P=0.001). 22% (n=5) of HRD+ pts completed 6 cycles compared to 67% (n=75) of HRD- pts. Additional factors associated with decreased likelihood of completing 6 cycles included a higher pretreatment TF (AOR 0.69, 95% CI, 0.48-0.97, P=0.034) and prior taxane use (AOR 0.41, 95% CI, 0.18-0.91, P=0.028), but not pretreatment PSA ( P=0.574). Conclusions: Targeted panel sequencing with error suppression from ctDNA identified HRD+ in mCRPC pts with bone-predominant disease and low median TF at a similar frequency as reported from tissue. In our cohort, HRD+ was prognostic of early clinical progression with Ra-223. Further work is in progress to understand the association of other ctDNA-derived features, including assessment of genomic signatures and transcriptional binding sites, in the setting of Ra-223 therapy. DF/HCC IRB protocol 18-135.

Effect of Usnic Acid-Derived Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Used as Monotherapy or in Combination with Olaparib on Transplanted Tumors In Vivo

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that removes various adducts from the 3' end of DNA. Such adducts are formed by enzymes that introduce single-strand breaks in DNA during catalysis (for example, topoisomerase 1) and a number of anticancer drugs with different mechanisms of action. Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme that catalyzes posttranslational modification (PARylation) of various targets and thus controls many cell processes, including DNA repair. Tdp1 is a PARP1 target, and its PARylation attracts Tdp1 to the site of DNA damage. Olaparib is a PARP1 inhibitor used in clinical practice to treat homologous recombination-deficient tumors. Olaparib inhibits PARylation and, therefore, DNA repair. The Tdp1 inhibitor OL7-43 was used in combination with olaparib to increase the antitumor effect of the latter. Olaparib cytotoxicity was found to increase in the presence of OL7-43 in vitro. OL7-43 did not exert a sensitizing effect, but showed its own antitumor and antimetastatic effects in Lewis and Krebs-2 carcinoma models.