PARP Inhibitors Research Articles

DNA Repair and Replicative Stress Addiction in Neuroblastoma

Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system. Survival remains poor for the almost 40% of patients with high-risk NB. Targeted therapy options for high-risk NB are limited and single compound strategies often fail due to escape mechanisms, driven either by tumor heterogeneity or adaptive (epigenetic) responses or mutations. Novel NB therapeutic approaches rely increasingly on biomarker selected cohorts for phase I/II clinical trials. Parallel intensive research programs are needed to identify novel therapeutic vulnerabilities or drug targeting strategies and to further inform clinical trials and prioritize potent, less toxic combinations. While several effective chemotherapies work by increasing replication stress in cancer cells, recently, newer putatively less toxic small molecule-based approaches that directly target DNA damage response (DDR) pathway components such as ATR, CHK1 and PARP inhibitors are being evaluated in early phase trials for many cancers. NB sequencing studies have identified recurrent alterations (copy number and mutations) in many of these genes encoding critical DDR pathway proteins suggesting susceptibility to specific classes of DDR-targeting therapies. In this review, we summarize current data supporting the roles of DDR and replicative stress addiction in NB, including genetic alterations which impact DDR signaling pathways. Finally, we review the mechanisms, pre-clinical evidence and ongoing trials for drugs that target DDR-deficient and/or replication addicted NB.

Alternative lengthening of telomere-based immortalization renders H3G34R -mutant diffuse hemispheric glioma hypersensitive to PARP inhibitor combination regimens

Abstract Background Diffuse hemispheric glioma, H3G34R/V-mutant (DHG-H3G34) is characterized by poor prognosis and lack of effective treatment options. DHG-H3G34R further harbor deactivation of Alpha-Thalassemia/Mental Retardation Syndrome X-linked protein (ATRX; DHG-H3G34R_ATRX) suggesting a unique interaction of these two oncogenic alterations. In this study, we dissect their cell biological interplay, investigate the impact on telomere stabilization and, consequently, validate a targeted therapy approach. Methods We characterized patient-derived primary pediatric high-grade glioma (pHGG) models for telomere-maintenance mechanisms, DNA damage stress (including protein expression, pH2AX/Rad51 foci, cell-cycle arrest) and their sensitivity towards poly-ADP polymerase inhibitor (PARPi) combinations. Human induced pluripotent stem cells (iPSCs) were used for modelling the disease. The anticancer activity of PARPi combinations in vivo was studied in Chorioallantoic Membrane (CAM) and orthotopic in vivo experiments. Finally, we treated a DHG-H3G34R_ATRX patient with a PARPi combination therapy. Results We elaborate that alternative lengthening of telomeres (ALT) is a key characteristic of DHG-H3G34R_ATRX. A dominant cooperative effect between H3G34R and ATRX loss in ALT activation also became apparent in iPSCs, which endogenously exert telomerase activity. In both, patient-derived DHG-H3G34R_ATRX models and H3G34R+/ATRX- iPSCs, the ALT phenotype was associated with increased basal DNA damage stress, mediating synergistic susceptibility towards PARPi (talazoparib, niraparib) combinations with topoisomerase-I inhibitors (topotecan, irinotecan). In a first-of-its-kind case, treatment of a DHG-H3G34R_ATRX patient with the brain-penetrant PARP inhibitor niraparib and topotecan resulted in a significant tumor reduction. Conclusion Our preclinical and clinical data strongly support the further development of PARPis together with DNA damage stress-inducing treatment regimens for DHG-H3G34R_ATRX.

Understanding tumour growth variability in breast cancer xenograft models identifies PARP inhibition resistance biomarkers

Understanding tumour growth variability in breast cancer xenograft models identifies PARP inhibition resistance biomarkers

Abstract C046: PARP inhibitors as immune modulators in metastatic ovarian cancer treatment

Abstract Epithelial ovarian carcinoma (EOC) is one of the top five causes of cancer-related death in women. Most patients with EOC achieve initial remission after primary or interval cytoreductive surgery combined with platinum-taxane chemotherapy. However, mutations in BRCA1 or BRCA2 genes, which lead to homologous recombination (HR) defects, play a crucial role in platinum sensitivity and justify the use of poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy, commonly linked to extended progression-free survival (PFS). Besides their direct cytotoxic and cytostatic effects, PARP inhibitors (PARPi) have shown significant immunostimulatory properties by disrupting DNA repair in cancer cells, and opening possibilities for synergy with immune checkpoint inhibitors (ICIs). In this study, we investigate the immunomodulatory effects of PARPi using multiparametric flow cytometry, multiplexed immunolabeling, single-cell transcriptomics, and functional assays in an experimental BR5Brca1-/- syngeneic mouse model and human EOC tumor samples. We examine the molecular and cellular mechanisms that can be exploited to develop more effective combination therapies. PARPi may increase the mutational burden in EOCs due to unresolved DNA damage and the release of damage-associated molecular patterns (DAMPs), thereby increasing T-cell infiltration. In addition, PARPi appear to promote potent type I interferon (IFN) secretion through the activation of cGAMP synthase and the stimulator of the interferon genes (STING) pathway. In combination with ICIs, PARPi showed a beneficial effect on the balance between adaptive anti-tumor immunity and innate myeloid components, leading to an improved cytotoxic T-cell response, as observed in mouse models and HGSOC tumor samples. These observations emphasize the role of strategically designed combinations of PARPi and immunotherapeutic agents, which could be the key to overcoming immunosuppression in the EOC microenvironment, thereby improving clinical outcomes. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Michal Hensler, Romana Mikyskova, Lenka Kasikova, Josef Pasuvka, Jana Drozenova, Katerina Mojzisova, Marek Kovar, Iain McNiesh, Michael Halaska, Lukas Rob, Sandra Orsulic, Milan Reinis, Lorenzo Galluzzi, Radek Spisek, Jitka Palich Fucikova. PARP inhibitors as immune modulators in metastatic ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C046.

USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity.

PARP inhibitors (PARPi) represent a game-changing treatment for patients with ovarian cancer with tumors deficient for the homologous recombination (HR) pathway treated with platinum (Pt)-based therapy. PARPi exert their cytotoxic effect by both trapping PARP1 on the damaged DNA and by restraining its enzymatic activity (PARylation). How PARP1 is recruited and trapped at the DNA damage sites and how resistance to PARPi could be overcome are still matters of investigation. Here, we described PARP1 as a substrate of the deubiquitinase USP1. At molecular level, USP1 binds PARP1 to remove its K63-linked polyubiquitination and controls PARP1 chromatin trapping and PARylation activity, regulating sensitivity to PARPi. In both Pt/PARPi-sensitive and -resistant cells, USP1/PARP1 combined blockade enhances replicative stress, DNA damage, and cell death. Our work dissected the biological interaction between USP1 and PARP1 and recommended this axis as a promising and powerful therapeutic choice for not only sensitive but also chemoresistant patients with ovarian cancer irrespective of their HR status.

PARG inhibitor sensitivity correlates with accumulation of single-stranded DNA gaps in preclinical models of ovarian cancer

Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA-damaging agents. PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as cancer patients with germline or somatic BRCA1/2-mutations. PARP inhibitors kill BRCA-deficient cancer cells by increasing single-stranded DNA gaps (ssGAPs) during replication. Here, we report that, like PARP inhibitor (PARPi), PARG inhibitor (PARGi) treatment also causes an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-phase-specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands and induced ssGAPs, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication forks and at the ssDNA sites in sensitive cells. Additionally, PARGi exhibited monotherapy activity in specific HR-deficient, as well as HR-proficient, patient-derived, or patient-derived xenograft (PDX)-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssGAPs. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssGAPs due to OFP defects during replication. Regardless of the BRCA/HRD-status, the induction of ssGAPs in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids (PDOs) may be a useful model system for testing PARGi sensitivity and functional biomarkers.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

Leveraging homologous recombination deficiency for sarcoma : Unravelling homologous recombination repair deficiency and therapeutic opportunities in soft tissue and bone sarcoma.

Homologous recombination deficiency (HRD) in tumors correlates with poor prognosis and metastases development. Determining HRD is of major clinical relevance as it can be treated with PARP inhibitors (PARPi). HRD remains poorly investigated in sarcoma, arare and heterogeneous cancer of mesenchymal origin. We aimed (i)to investigate predictive biomarkers of HRD in several independent sarcoma cohorts using across-functional strategy by combining genomic, transcriptomic and phenotypic approaches and (ii)to evaluate the therapeutic potential of PARPi and DNA damage response (DDR)-based therapies ex vivo. We performed acomprehensive genomic and transcriptomic characterization of sarcoma using datasets from The Cancer Genome Atlas (TCGA) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and our own bone and soft tissue sarcoma cohorts. We evaluated PARP1/2 and WEE1 inhibition ex vivo in patient-derived sarcoma cell models as monotherapy and in combination with chemotherapeutic agents to identify synergistic effects. Firstly, we identified genomic traits of HRD in asubset of sarcomas associated with molecular alterations in homologous recombination repair (HRR) pathway genes and high chromosomal instability. Secondly, we identified and validated distinct SARC-HRD transcriptional signatures that predicted sensitivity to PARPi. Finally, we showed functional defects in HRR in sarcoma cells that were associated with functional dependency towards PARPi and WEE1i and support the clinical use of RAD51 as apredictive biomarker for PARPi sensitivity. We provide apersonalized oncological approach to potentially improve the treatment of sarcoma patients. We encourage the evaluation of gene expression signatures to enhance the identification of patients who might benefit from DDR-based therapies.

Hyperthermic intraperitoneal chemotherapy for recurrent ovarian cancer (CHIPOR): a randomised, open-label, phase 3 trial.

Hyperthermic intraperitoneal chemotherapy (HIPEC) at interval cytoreductive surgery for ovarian cancer improves overall survival but its role in recurrent disease is uncertain. We aimed to compare outcomes in patients treated with or without HIPEC during surgery for recurrent ovarian cancer. The multicentre, open-label, randomised, phase 3 CHIPOR trial was conducted at 31 sites in France, Belgium, Spain, and Canada, and enrolled patients with first relapse of epithelial ovarian cancer at least 6 months after completing platinum-based chemotherapy. Eligible patients were aged 18 years or older with WHO performance status of less than 2. After six cycles of platinum-based chemotherapy (and optional bevacizumab), patients amenable to complete cytoreductive surgery were randomly assigned centrally in a 1:1 ratio, using a web-based system and a minimisation procedure, during surgery to receive HIPEC (cisplatin 75 mg/m2 in 2 L/m2 of serum at 41±1°C for 60 min) or not, stratified by centre, completeness of cytoreduction score, platinum-free interval, and latterly, planned poly(ADP-ribose) polymerase inhibitor use. The primary endpoint was overall survival, analysed on an intention-to-treat basis in all randomly assigned patients. This ongoing trial is registered with ClinicalTrials.gov, NCT01376752. Between May 11, 2011, and May 14, 2021, 415 female patients were randomly assigned (207 HIPEC, 208 no HIPEC). At the primary analysis (median follow-up 6·2 years, IQR 4·1-8·1), 268 (65%) patients had died (126 [61%] of 207 in the HIPEC group; 142 [68%] of 208 in the no-HIPEC group). Overall survival was significantly improved with HIPEC (stratified hazard ratio 0·73, 95% CI 0·56-0·96; p=0·024). Median overall survival was 54·3 months (95% CI 41·9-61·7) with HIPEC versus 45·8 months (38·9-54·2) without. Grade 3 or worse adverse events within 60 days after surgery occurred in 102 (49%) of 207 patients receiving HIPEC versus 56 (27%) of 208 receiving no HIPEC, the most common being anaemia (47 [23%] vs 30 [14%]), hepatotoxicity (23 [11%] vs 18 [9%]), electrolyte disturbance (28 [14%] vs two [1%]), and renal failure (20 [10%] vs three [1%]). There were three deaths within 60 days of surgery, all in the no-HIPEC group. Adding HIPEC to cytoreductive surgery after response to platinum-based chemotherapy at first epithelial ovarian cancer recurrence significantly improved overall survival. When treating patients with late first relapse of high-grade serous or high-grade endometrioid ovarian cancer amenable to complete cytoreductive surgery at specialist centres, platinum-based HIPEC should be considered to extend overall survival. French National Cancer Institute and French League Against Cancer.

DNA damage response inhibitors in cancer therapy: lessons from the past, current status and future implications.

The DNA damage response (DDR) is a network of proteins that coordinate DNA repair and cell-cycle checkpoints to prevent damage being transmitted to daughter cells. DDR defects lead to genomic instability, which enables tumour development, but they also create vulnerabilities that can be used for cancer therapy. Historically, this vulnerability has been taken advantage of using DNA-damaging cytotoxic drugs and radiotherapy, which are more toxic to tumour cells than to normal tissues. However, the discovery of the unique sensitivity of tumours defective in the homologous recombination DNA repair pathway to PARP inhibition led to the approval of six PARP inhibitors worldwide and to a focus on making use of DDR defects through the development of other DDR-targeting drugs. Here, we analyse the lessons learnt from PARP inhibitor development and how these may be applied to new targets to maximize success. We explore why, despite so much research, no other DDR inhibitor class has been approved, and only a handful have advanced to later-stage clinical trials. We discuss why more reliable predictive biomarkers are needed, explore study design from past and current trials, and suggest alternative models for monotherapy and combination studies. Targeting multiple DDR pathways simultaneously and potential combinations with anti-angiogenic agents or immune checkpoint inhibitors are also discussed.

Metabolically inducing defects in DNA repair sensitizes BRCA-wild-type cancer cells to replication stress.

Metabolic reprogramming from oxidative respiration to glycolysis is generally considered to be advantageous for tumor initiation and progression. However, we found that breast cancer cells forced to perform glycolysis acquired a vulnerability to PARP inhibitors. Small-molecule inhibition of mitochondrial respiration-using glyceollin I, metformin, or phenformin-induced overproduction of the oncometabolite lactate, which acidified the extracellular milieu and repressed the expression of homologous recombination (HR)-associated DNA repair genes. These serial events created so-called "BRCAness," in which cells exhibit an HR deficiency phenotype despite lacking germline mutations in HR genes such as BRCA1 and BRCA2, and, thus, sensitized the cancer cells to clinically available poly(ADP-ribose) polymerase inhibitors. The increase in lactate repressed HR-associated gene expression by decreasing histone acetylation. These effects were selective to breast cancer cells; normal epithelial cells retained HR proficiency and cell viability. These mechanistic insights into the BRCAness-prone properties of breast cancer cells support the therapeutic utility and cancer cell-specific potential of mitochondria-targeting drugs.

CTIM-32. PHASE I/II STUDY OF STEREOTACTIC RADIOSURGERY WITH CONCURRENT OLAPARIB FOLLOWED BY ADJUVANT DURVALUMAB AND PHYSICIAN’S CHOICE SYSTEMIC THERAPY IN SUBJECTS WITH BREAST CANCER BRAIN METASTASES (SOLARA)

Abstract BACKGROUND Despite progress in the treatment of brain metastasis for HER2+ breast cancer, outcomes for patients with HER2-negative breast cancer brain metastases remain poor. Preclinical studies show inhibitors of poly(ADP-ribose) polymerase (PARP) are effective in combination with radiation therapy as a DNA damage response inhibitor. Triple-negative breast cancer (TNBC) has higher rates of homologous recombination deficiency compared to other subtypes, and together with HER2-negative, BRCA-mutated breast cancer would be particularly sensitive to PARP inhibition. PARP inhibition has also demonstrated promising efficacy combined with immunotherapy in patients with germline BRCA-mutant and metastatic TNBC in clinical trials. In addition, immunotherapy with stereotactic radiosurgery (SRS) is associated with favorable intracranial control and survival in patients with brain metastases. We hypothesize that this biologically-driven combination will enhance local control of SRS-treated brain metastases through synergy with PARP inhibition, while controlling micrometastatic disease via potentiation of the immune response. METHODS We are conducting a multi-institution, Phase I/II trial of SRS plus olaparib, followed by durvalumab (with physician’s choice systemic therapy), for patients with TNBC (any BRCA status) or HER2-negative with BRCA-mutated (germline or somatic) breast cancer brain metastases [NCT04711824]. A total of 41 patients are planned for enrollment at 8 sites. The primary objectives are to evaluate safety and tolerability (Phase I) and determine intracranial disease control at 6 months (Phase II). Secondary objectives include assessing intracranial and global progression-free survival, overall survival, and intracranial and extracranial response rate. Exploratory objectives will assess potential biomarkers of treatment response, including changes in circulating tumor cells and DNA in blood and cerebrospinal fluid, germline and tumor mutations in DNA repair pathway genes, and PD-L1 expression, as well as quality of life and patient-reported outcomes. As of May 2024, phase I has been completed without dose-limiting toxicity, and phase II is currently enrolling. Funding/drug provision from AstraZeneca.

Olaparib-induced myelodysplasia – A case report

Olaparib is an antineoplastic agent that is a poly ADP ribose polymerase (PARP) inhibitor and is Food and Drug Administration-approved for the treatment of ovarian, breast, pancreatic, and prostate cancer. PARP inhibitors can cause DNA damage and epigenetic changes, leading to hematological transformations and increasing the risk of myelodysplasia. We present a case of a 58-year-old female with malignant breast cancer initiated on Olaparib after failing multiple treatment lines. The patient had persistent declining blood cell counts, and a bone marrow biopsy revealed hypercellular bone marrow with predominant myeloid cells with increasing number of blasts and promyelocytes. Olaparib was stopped. The patient elected for supportive care and hospice.

CTNI-50. CLINICAL EFFICACY AND PK/PD RESPONSE IN A PHASE 0/2 STUDY OF NIRAPARIB PLUS RADIOTHERAPY FOR NEWLY-DIAGNOSED, MGMT-UNMETHYLATED GLIOBLASTOMA

Abstract BACKGROUND Poly (ADP-ribose) polymerase (PARP) mediates DNA damage response; niraparib is a PARP1/2-selective inhibitor. This Phase 0/2 study evaluates tumor pharmacokinetics (PK), pharmacodynamics (PD), and clinical efficacy of niraparib plus fractionated radiotherapy in newly diagnosed, O6-methylguanine methyltransferase (MGMT)-unmethylated glioblastoma (GBM). METHODS Newly-diagnosed GBM patients received 4 days of niraparib (300/200 mg QD) prior to planned resection 3-5 (cohort 1) or 8-10 hours (cohort 2) following the last dose. Tumor tissue (gadolinium-enhancing and non-enhancing regions), cerebrospinal fluid (CSF), and plasma were collected. Total and unbound drug concentrations were measured using validated LC-MS/MS methods. PARP inhibition was assessed by quantification of PAR induction after ex vivo irradiation vs non-irradiated control. A PK response was defined as unbound [niraparib] > 5-fold biochemical IC50 (19 nM) in non-enhancing tumor. Patients with MGMT-unmethylated tumors and a PK response were eligible for Phase 2 dosing of niraparib plus radiotherapy followed by niraparib monotherapy. RESULTS All Phase 0 patients (n=46) met the PK threshold. The mean unbound concentrations of niraparib in non-enhancing tumor regions were 335.1 nM (cohort 1; n=43) and 331.9 nM (cohort 2; n=3). Ex vivo PAR suppression was observed in 73% of the patients (24/33). 71.4% patients with unmethylated tumors were enrolled into Phase 2. Five patients in Phase 2 experienced Grade 4 thrombocytopenia related to niraparib and all adverse events resolved without sequelae. At a mean clinical follow-up of 13.4 months, median overall survival was 20.3 months. CONCLUSIONS Niraparib achieves pharmacologically relevant concentrations in non-enhancing, newly-diagnosed GBM tissue. Accompanying PD effects were observed in tumor tissue, no new safety signals were observed, and clinical outcomes were promising. A global Phase 3, open-label, randomized 2-arm study comparing niraparib versus temozolomide in adult patients with newly diagnosed, MGMT-unmethylated glioblastoma is underway.

Olaparib enhancing radiosensitization and anti-metastatic effect of oral cancer by targeting IL-17A signal.

We tested whether the PARP inhibitor, Olaparib, can effectively enhance radiosensitivity while inhibiting OSCC growth and metastasis in vitro and in vivo. Patient samples were used for survival validation. The present study investigated the effect of Olaparib and ionizing radiation (IR) on clonogenic, migratory, and invasive ability in human IR-sensitive (OML1) and IR-resistant (OML1-R) OSCC cell lines. We next explored the underlying mechanism with ELISA and a Western blotting assay. Two in vivo mouse models were established to investigate the efficacy of Olaparib combined with radiotherapy (RT) on local tumor growth and lung metastasis. IL-17A expression was confirmed in tissue specimens of OSCC patients by immunohistochemistry. We found that Olaparib, in combination with IR, substantially inhibited cell growth, migration, and invasion in vitro. Mechanistically, the Olaparib treatment significantly reduced the secretion of IL-17A in irradiated OSCC cells by attenuating NF-κB and p38 activity. Consistently, Olaparib enhanced the radiosensitivity and, with RT, synergistically reduced both tumor growth and lung metastasis in mice. In addition, OSCC patients with high IL-17A expression were substantially associated with an increased risk of lymph node involvement and worse survival. This study has highlighted that Olaparib displays radiosensitizing and antimetastatic effects by inhibiting the IL-17A-dependent signal. Remarkably, Olaparib could provide a remarkable anticancer efficacy to improve treatment response in OSCC patients with recurrent/metastatic disease after RT.

DDDR-15. UTILIZING DEEP DOCKING AND ARTIFICIAL INTELLIGENCE FOR THE DISCOVERY OF NOVEL PARP1-SELECTIVE INHIBITORS FOR USE AGAINST BRAIN TUMORS

Abstract Brain tumors, including primary brain tumors and central nervous system (CNS) metastases, remain among the most challenging malignancies to treat, with therapeutic options often limited by the inability of drugs to penetrate the blood-brain barrier. Poly(ADP-ribose) polymerase (PARP) is a key enzyme in DNA repair, and inhibition of PARP1 specifically drives synthetic lethality in BRCA-mutated disease. While they’ve achieved commercial success, first generation PARP inhibitors are limited in their utility as they cannot readily pass through the blood-brain barrier and have adverse side effects, likely driven by the collateral inhibition of PARP2. Development of a PARP1-selective, CNS penetrant inhibitor could reduce toxicity, while providing a new therapeutic option for brain tumors. Traditional drug discovery methods are time-consuming and costly, necessitating innovative approaches. Here, we describe the application of Deep Docking, an advanced artificial intelligence (AI) approach, to discover a novel, PARP1-selective inhibitor for use against brain tumors. Deep Docking utilizes deep learning to accelerate the prediction of the binding affinity of a large number of compounds to target proteins, streamlining the virtual screening process and allowing for rapid docking of billions of compounds against the PARP1 protein. Additionally, state-of-the-art generative algorithms and deep learning techniques for predicting drug-like properties such as metabolism, permeability, and safety profiles, can be combined to rapidly perform hit-to-lead optimization. We will present preliminary Deep Docking screening results from billions of compounds, identifying several with predicted high binding affinities for PARP-1. Validating in vitro and in vivo data, including PARP1 selectivity, metabolic stability, pharmacokinetic profile, CNS penetration and safety profile, will be described. Application of the Deep Docking AI platform is being used to significantly accelerate the discovery of a selective PARP-1 inhibitor for brain tumors. This approach will not only improve the efficiency of drug discovery but also enhance the specificity and efficacy of potential therapeutics.

BIOM-66. GENOME-WIDE DNA METHYLATION PATTERNS IN PERIPHERAL BLOOD OF PATIENTS WITH GLIOBLASTOMA ENROLLED ON THE PHASE II VERTU TRIAL

Abstract BACKGROUND For patients with glioblastoma, the identification of a non-invasive biomarker for longitudinal molecular profiling could improve risk stratification, clinical trial design, and surveillance. In a secondary analysis of the phase II randomized VERTU trial (ACTRN12615000407594) of adult patients with newly diagnosed MGMT-unmethylated glioblastoma, we assessed the feasibility of characterizing peripheral blood genome-wide DNA methylation patterns. METHODS DNA was extracted from whole blood and serum samples at baseline and end-of-treatment (EOT) from patients enrolled on VERTU, which evaluated the PARP inhibitor veliparib combined with radiotherapy and temozolomide. Infinium FFPE QC qPCR and DNA Restoration Kits were used to restore and concentrate fragmented serum DNA. Genomic DNA was plated, bisulfite converted, and profiled for methylation patterns at >935,000 CpG sites using the Infinium MethylationEPIC microarray V2.0. Quality control, pre-processing, leukocyte deconvolution, and calling of MGMT status were completed in R Studio. RESULTS Peripheral blood methylation data were generated and passed quality control for 104/104 baseline and 24/34 EOT samples available for analysis. The median concentration of genomic DNA at baseline and EOT was 104.50 ng/μl (range:1.97-550) and 0.98 ng/μl (range:0.11-19.8). The deconvoluted median leukocyte proportions differed significantly between baseline and EOT for neutrophils (0.64vs0.90,p<0.01), CD4 memory (0.09vs0.00,p<0.01), CD4 naive (0.03vs0.01,p=0.02), CD8 memory (0.04vs0.00,p<0.01), monocyte (0.07vs0.03,p<0.01), natural killer (0.05vs0.00,p<0.01), and T regulatory (0.000vs0.001,p=0.01) cells. There were no differences in baseline leukocyte proportions between patients with (n=6) and without (n=98) biopsy-proven pseudoprogression. 2/104 samples were determined to be MGMT-methylated based on peripheral blood methylation analysis. Unsupervised hierarchical clustering based on the 1,000 most differentially methylated CpG sites generated two patient clusters (n=64, n=40). CONCLUSIONS To our knowledge, this is the first study to demonstrate the feasibility of analyzing peripheral blood methylation patterns from patients with glioblastoma receiving chemoradiation on a randomized clinical trial. Efforts are underway to correlate these blood methylation patterns with previously published tumor tissue methylation patterns and clinical outcomes.

Enhancing low-dose radiotherapy efficacy with PARP inhibitors via FBL-mediated oxidative stress response in colorectal cancer.

The effectiveness of radiotherapy in colorectal cancer (CRC) relies on its ability to induce cell death via the generation of reactive oxygen species (ROS). However, genes responsible for mitigating oxidative stress can impede radiotherapy's efficacy. In this study, we elucidate a significant association between the nucleolar protein Fibrillarin (FBL) and the oxidative stress response in CRC tumors. Our findings reveal elevated expression of FBL in colorectal cancer, which positively correlates with oxidative stress levels. Mechanistically, FBL demonstrates direct accumulation at DNA damage sites under the regulation of PARP1. Specifically, the N-terminal GAR domain of FBL is susceptible to PARylation by PARP1, enabling FBL to recognize PARylated proteins. The accumulation of damaged FBL plays a pivotal role in facilitating short-patched base excision repair by recruiting Ligase III and disassociating PCNA and FEN1. Moreover, tumors with heightened FBL expression exhibit reduced DNA damage levels but increased sensitivity to combined low-dose radiotherapy and olaparib treatment. This underscores the potential of leveraging PARP inhibitors to augment radiotherapy sensitivity in CRC cases characterized by elevated FBL expression, offering a promising therapeutic avenue.

PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

Efficient DNA double strand break (DSB) repair is necessary for genomic stability and determines efficacy of DNA damaging cancer therapeutics. Spatiotemporal dynamics and post-translational modifications of repair proteins at DSBs dictate repair efficacy. Here, we identified a non-canonical function of GCN5 in regulating both HR and NHEJ repair post genotoxic stress. Mechanistically, genotoxic stress induced GCN5 recruitment to DSBs. GCN5 PARylation by PARP1 was essential for its recruitment, acetyltransferase activity and DSB repair function. Liquid chromatography-mass spectrometry (LC–MS) identified DNA-PKcs as part of GCN5 interactome. In-vitro acetyltransferase assays revealed that GCN5 acetylates DNA-PKcs at K3241 residue, a prerequisite for DNA-PKcs S2056 phosphorylation and DSB recruitment. Alongside, ChIP-qPCR revealed GCN5 mediates transcription of PRKDC via H3K27Ac acetylation in its promoter region (− 710 to − 554). Genetic perturbation of GCN5 also decreased CHEK1, NBN1, TP53BP1, POL-L transcription and abrogated ATM, BRCA1 activation. Accordingly, GCN5 loss led to persistent ɣ-H2AX foci formation, compromised in-vivo HR-NHEJ and caused GBM radio-sensitization. Importantly, PARP1 inhibition phenocopied GCN5 loss. Together, this study identifies an untraversed DSB repair function of GCN5 and provides mechanistic insights into transcriptional as well as post-translational regulation of pivotal HR-NHEJ factors. Alongside, it highlights the translational importance of PARP1-GCN5 axis in mediating GBM radio-resistance.

DNA damage-induced EMT controlled by the PARP-dependent chromatin remodeler ALC1 promotes DNA repair efficiency through RAD51 in tumor cells.

Epithelial-to-mesenchymal transition (EMT) allows cancer cells to metastasize while acquiring resistance to apoptosis and chemotherapeutic agents with significant implications for patients' prognosis and survival. Despite its clinical relevance, the mechanisms initiating EMT during cancer progression remain poorly understood. We demonstrate that DNA damage triggers EMT and that activation of poly (ADP-ribose) polymerase (PARP) and the PARP-dependent chromatin remodeler ALC1 (CHD1L) was required for this response. Our results suggest that this activation directly facilitates access to the chromatin of EMT transcriptional factors (TFs) which then initiate cell reprogramming. We also show that EMT-TFs bind to the RAD51 promoter to stimulate its expression and to promote DNA repair by homologous recombination. Importantly, a clinically relevant PARP inhibitor reversed or prevented EMT in response to DNA damage while resensitizing tumor cells to other genotoxic agents. Overall, our observations shed light on the intricate relationship between EMT, DNA damage response, and PARP inhibitors, providing potential insights for in cancer therapeutics.