Homologous Recombination DNA Research Articles

DNA damage response mutations enhance the antitumor efficacy of ATR and PARP inhibitors in cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA) is a biliary tract carcinoma that is challenging to treat due to its heterogeneity and limited treatment options. Genetic alterations in DNA damage response (DDR) pathways and homologous recombination (HR) defects are common in CCA. This has prompted interest in the use of ataxia telangiectasia and Rad3-related protein (ATR) and poly(ADP-ribose) polymerase (PARP) inhibitors to treat CCA. The present study investigated the impact of an ATR inhibitor and various PARP inhibitors, individually and in combination, on CCA cell lines with different DDR mutation profiles. DDR gene alterations in these cell lines were analyzed, and the responses of the cells to treatment with the PARP inhibitors olaparib, veliparib and talazoparib and/or the ATR inhibitor AZD6738 were evaluated. Assessments focused on cellular viability, clonogenic survival and the combination index, alongside changes in DNA damage assessed via the formation of micronuclei and γ-H2A histone family member X foci. The results revealed that the CCA cell lines with more DDR mutations exhibited greater sensitivity to single and combination treatments. Talazoparib was found to be the most potent PARP inhibitor in the CCA cell lines. The combination of AZD6738 and talazoparib demonstrated varying synergistic effects depending on the genetic background of the CCA cells, with greater efficacy in the cell lines less sensitive to single drug treatments. Mechanistically, this combination promoted the accumulation of DNA damage, including DNA double-strand breaks. Overall, the study underscores the importance of HR in CCA. It reveals an association between the extent of DDR mutations and the response to AZD6738 and PARP inhibitors in CCA, both as single agents and in combination. These findings highlight that the number of mutated genes influences variability in the drug response.

Transcriptional analysis reveals the suppression of RAD51 and disruption of the homologous recombination pathway during PEDV infection in IPEC-J2 cells

PEDV is a highly contagious enteric pathogen that can cause severe diarrhea and death in neonatal pigs. Despite extensive research, the molecular mechanisms of host’s response to PEDV infection remain unclear. In this study, differentially expressed genes (DEGs), time-specific coexpression modules, and key regulatory genes associated with PEDV infection were identified. The analysis revealed 2,275, 1,492, and 3,409 DEGs in infected vs. mock-treated pigs at 12 h, 24 h, and 48 h, respectively. Time series analysis revealed that the upregulated genes were involved mainly in antiviral pathways such as the viral defense response and the regulation of immune system processes. Protein–protein interaction network analysis identified the top 20 core genes in the interaction network, which included six upregulated genes (TFRC, SUOX, RMI1, CD74, IFIH1, and CD86) and 14 downregulated genes (FOS, CDC6, CDCA3, PIK3R2, TUFM, VARS, ASF1B, POLD1, MCM8, POLA1, CDC45, BCS1L, RAD51, and RPA2). In addition, GSEA enrichment analysis revealed that pathways such as DNA replication and homologous recombination involving RAD51, CDC6, and RPA2 were significantly inhibited during viral infection. Our findings not only reveal dynamic changes in the transcriptome profile of PEDV-infected IPEC-J2 cells but also provide novel insights into the mechanism of PEDV infection of the host.

PARP Pioneers: Using BRCA1/2 Mutation-targeted Inhibition to Revolutionize Breast Cancer Treatment.

Breast Cancer stands on the second position in the world in being common and women happen to have it with high rate of about five-folds around the world. The causes of occurrence can matter with different humans be it external factors or the internal genetic ones. Breast cancer is primarily driven by mutations in the BRCA1 and BRCA2 susceptibility genes. These BC susceptibility genes encode proteins critical for DNA homologous recombination repair (HRR). Poly (ADP ribose) polymerases (PARP) are the essential enzymes involved in the repairing of the damaged DNA. So the inhibition of these inhibitors can be considered as the promising strategy for targeting cancers with defective damage in the deoxyribonucleic acid. Olaparib and talazoparib are PARP inhibitors (PARPi) are being employed for the monotherapies in case of the deleterious germline HER2-negative and BRCA-mutated breast cancer. The potency of PARP for trapping on DNA and causes cytotoxicity may have difference in the safety and efficacy with the PARPi. The PARPi have been found its place in the all different types of Breast Cancers and have shown potential benefits. The purpose of this review is to provide an update on the oral poly(ADP-ribose) polymerase (PARP)inhibitors for the improvement in the treatment and management of Breast Cancer.

Life table analysis and RNA-Seq reveal hormesis and transgenerational effects of deltamethrin on Aphis gossypii.

Deltamethrin, as a highly effective and broad-spectrum insecticide, has been widely used for agricultural pest control such as Aphis gossypii worldwide. Increasing evidence has shown that despite great economic benefits brought by it, deltamethrin has also non-negligible side effects. However, the potential risks and related molecular mechanisms remain largely unclear. Herein, the life table parameters and transcriptome sequencing analyses of the four successive aphid generations were performed to investigate the hormesis and transgenerational effects of deltamethrin on A. gossypii. The life table analysis showed that although the exposure of G0 aphid to 30% lethal concentration (LC30) deltamethrin significantly reduced the net reproduction rate (R0), intrinsic rate of increase (r), and fecundity of G0, but it significantly enhanced the R0 and fecundity of subsequent two generations (G1 and G2) of A. gossypii. Moreover, transcriptomic analyses showed that the signaling pathways related to posttranscriptional regulation (spliceosome), protein processing, longevity regulating, and cell proliferation (DNA replication, homologous recombination and non-homologous end-joining) were significantly up-regulated in G1 or G2 under LC30 deltamethrin treatment. Additionally, we also found that the deltamethrin-sulfoxaflor rotation of G0 and G1 still induced reproductive stimulation, but the reproductive stimulation induced by insecticides rotation treatment was significantly lower than that in the deltamethrin exposure alone. Our study demonstrates that sublethal concentrations of deltamethrin significantly enhanced the offspring fecundity of cotton aphid. In addition, our study also reveals the transcriptional response mechanism of hormesis-induced fecundity increase, providing valuable reference for optimizing the application of deltamethrin in integrated pest management. © 2024 Society of Chemical Industry.

Delivery of PARP inhibitors through 2HG-incorporated liposomes for synergistically targeting DNA repair in cancer

PARP inhibitors (PARPi) benefit only a small subset of patients with DNA homologous recombination (HR) defects. In addition, long-term administration of a PARPi can lead to the development of drug resistance. 2-Hydroxyglutarate (2HG) has long been known as an oncometabolite but is capable of inducing an HR defect, which makes tumor cells exquisitely sensitive to PARPi. To facilitate the translation of this discovery to the treatment of both HR-deficient and HR-proficient tumors, a liposomal formulation was developed for codelivery of 2HG and veliparib, a PARPi. A sequential loading protocol was developed such that the initial loading of 2HG into liposomes greatly facilitated the subsequent, pH gradient-driven remote loading of veliparib. The liposomes co-loaded with veliparib and 2HG exhibited favorable stability, slow kinetics of drug release, and targeted delivery to the tumor. Furthermore, the veliparib/2HG liposomes demonstrated enhanced anti-tumor activity in both PARPi-resistant BRCA mutant cancer and BRCA wildtype cancer by synergistically enhancing the defect in DNA repair. Moreover, combination of veliparib and 2HG via liposomal co-delivery also augmented the function of cytotoxic T cells by activating the STING pathway and downregulating PD-L1 expression via 2HG-induced hypermethylation.

Genomic Landscape of Osteosarcoma of Bone in an Older-Aged Patient Population and Analysis of Possible Etiologies Based on Molecular Signature.

Background: Osteosarcoma (OS), the most common primary malignant bone tumor, occurs mostly in the pediatric and adolescent (P/A) population where it has been subject to intense study whereas OS arising in the older-aged adult population has undergone less scrutiny. Materials and Methods: In this study, we assess the molecular aberrations detected in eight older adult patients (>59 years of age) with OS of bone by whole-exome sequencing (WES) on formalin-fixed, paraffin-embedded tissue and quantified the contributions of endogenous and exogenous mutational processes to tumor mutational burden and to tumorigenesis through computational analysis. Results: We identified 86 clinically significant somatic mutations. TP53 mutations occurred in OSs of three patients and one patient harbored a pathogenic germline mutation of TP53. Loss-of-heterozygosity of DNA-damage repair genes occurred in all six tumors evaluated. Computational analysis of single nucleotide variants within each tumor detected eight distinct mutagenic processes of which age-associated mutational processes, thiopurine chemotherapy, and defective homologous DNA recombination repair contributed the most to both tumor mutation burden and tumor pathogenesis. Conclusion: The genomic landscape of our older OS patients deciphered by WES is extremely diverse with only 15% of mutated somatic genes uncovered in our study previously described in P/A-enriched OS studies. Endogenous age-related mutagenic processes, defective DNA homologous recombination repair, and exogenous effects of chemotherapy are mainly responsible for pathogenic mutations in OS occurring in our cohort.

53BP1 loss elicits cGAS-STING-dependent antitumor immunity in ovarian and pancreatic cancer

53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks, thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the ‘leaky’ DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic double-stranded DNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer and pancreatic ductal adenocarcinoma, which are refractory to ICBs, reveals that lower 53BP1 levels correlate with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

Machine learning-driven mast cell gene signatures for prognostic and therapeutic prediction in prostate cancer

BackgroundThe role of Mast cells has not been thoroughly explored in the context of prostate cancer's (PCA) unpredictable prognosis and mixed immunotherapy outcomes. Our research aims to employs a comprehensive computational methodology to evaluate Mast cell marker gene signatures (MCMGS) derived from a global cohort of 1091 PCA patients. This approach is designed to identify a robust biomarker to assist in prognosis and predicting responses to immunotherapy. MethodsThis study initially identified mast cell-associated biomarkers from prostate adenocarcinoma (PRAD) patients across six international cohorts. We employed a variety of machine learning techniques, including Random Forest, Support Vector Machine (SVM), Lasso regression, and the Cox Proportional Hazards Model, to develop an effective MCMGS from candidate genes. Subsequently, an immunological assessment of MCMGS was conducted to provide new insights into the evaluation of immunotherapy responses and prognostic assessments. Additionally, we utilized Gene Set Enrichment Analysis (GSEA) and pathway analysis to explore the biological pathways and mechanisms associated with MCMGS. ResultsMCMGS incorporated 13 marker genes and was successful in segregating patients into distinct high- and low-risk categories. Prognostic efficacy was confirmed by survival analysis incorporating MCMGS scores, alongside clinical parameters such as age, T stage, and Gleason scores. High MCMGS scores were correlated with upregulated pathways in fatty acid metabolism and β-alanine metabolism, while low scores correlated with DNA repair mechanisms, homologous recombination, and cell cycle progression. Patients classified as low-risk displayed increased sensitivity to drugs, indicating the utility of MCMGS in forecasting responses to immune checkpoint inhibitors. ConclusionThe combination of MCMGS with a robust machine learning methodology demonstrates considerable promise in guiding personalized risk stratification and informing therapeutic decisions for patients with PCA.

Abstract A019: Inhibition of nicotinamide adenine dinucleotide (NAD) production is a potent therapeutic strategy to inactivate homologous recombination in cancer cells

Abstract Homologous recombination (HR) is the most faithful DNA double-strand breaks (DSBs) repairing pathway. HR deficiency (HRD) can result from mutations in BRCA1, BRCA2, and PALB2, which are associated with genomic instability and cancer. HR deficient cells can survive through the use of alternative DNA repair pathways. Therefore, inhibiting these repair pathways in HRD cells trigger cancer cell death. This phenomenon is called synthetic lethality. The best-known example of synthetic lethality is that involving BRCA1/BRCA2/PALB2 deficiency with PARP-1 inhibition by Olaparib. However, ∼40% of patients will develop resistance to PARP inhibition because of several mechanisms, including a second mutation in BRCA1/2, restoring their full-length expression or RAD51 overexpression. RAD51 is one of the most important mediators of HR as it promotes the invasion of a sister chromatid allowing faithful DSB repair. There are many pieces of evidence which show that RAD51 overexpression is correlated with resistance and worse overall survival. Our hypothesis is that targeting RAD51 can trigger HR deficiency, particularly in cancer cells resistant to standard treatments. We thus sought to find inhibitors of the RAD51 recombinase. We have developed an in cellulo screening technique to test the direct effect of chemical compounds on RAD51 foci formation following irradiation. Using this technique, we identified , among 1381 chemical molecules, FK866 (Daporinad) which is a Nicotinamide Phosphoribosyltransferase Inhibitor (NAMPTi). NAMPT is involved in producing cellular Nicotinamide Adenine Dinucleotide (NAD+), which plays a crucial role in various metabolic processes within the cell, primarily in energy production and DNA repair. Remarkably, treatment of Daporinad reduced RAD51 foci formation and triggered a destabilization of the RAD51 protein by the proteasome. This destabilization seemed to be specific for RAD51, as NHEJ factors were not affected by NAMPTi. We will present data related to our objectives: i) Monitoring the effect of Daporinad on different cancer cell lines originated from cancer types which has shown resistance and worse overall survival because of RAD51 overexpression. Western blot and qPCR techniques will be used to investigate the effect of Daporinad on RAD51 protein and mRNA level and using Zeiss Celldiscoverer 7 system and Incucyte live cell imaging microscopy to observe the effect of Daporinad on cell survival; ii) To confirm the specific targeting of HR by Daporinad, we will monitor the effect of Daporinad on HR and other DNA DSB repair pathways using in cellulo reporter systems; iii) In the long term, we aim to evaluate the effect of Daporinad in vivo on tumor cell growth in pre-clinical models, such as mouse xenografts. In conclusion, small molecules affecting HR provide a source of new cancer therapies targeting RAD51 overexpression or using synthetic lethality. Citation Format: Sadaf Valeh Sheida, Thibaut Peterlini, Mélissa Thomas, Guy Poirier, Jean-Yves Masson. Inhibition of nicotinamide adenine dinucleotide (NAD) production is a potent therapeutic strategy to inactivate homologous recombination in cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A019.

The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity.

The clinical success of PARP1/2 inhibitors (PARPi) prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARPi. We show that the sensitivity of POLE4 knockouts is not due to compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 loss affects replication speed leading to the accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment, due to impaired post-replicative repair. POLE4 knockouts elicit elevated replication stress signaling involving ATR and DNA-PK. We find POLE4 to act parallel to BRCA1 in inducing sensitivity to PARPi and counteracts acquired resistance associated with restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARPi driven therapies and hamper acquired PARPi resistance.

RAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

Iron promotes ovarian cancer malignancy and advances platinum resistance by enhancing DNA repair via FTH1/FTL/POLQ/RAD51 axis

Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of ‘Iron addiction’, an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.

RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.

Abstract PO1-06-09: Phase 2 Trial with Safety Run-In of Gedatolisib Plus Talazoparib in Advanced Triple Negative or BRCA1/2 Positive, HER2 Negative Breast CancersBig Ten Cancer Research Consortium BTCRC-BRE18-337

Abstract Up to 2/3 of triple negative breast cancers (TNBC) have acquired defects in homologous recombination (HR) DNA repair, yet poly (ADP-ribose) polymerase inhibitor (PARPi) monotherapy has been largely ineffective in the absence of a germline BRCA 1/2 mutation (gBRCA1/2). Phosphoinositide-3-kinase (PI3K)/mTOR pathway alterations are also common in breast cancers. Preclinical data suggest that PI3K/mTOR inhibition may disrupt normal function of the HR complex and increase dependency on PARP enzymes for HR DNA repair. Thus, combining a PI3K/mTOR inhibitor with a PARPi may result in a synergistic anti-neoplastic effect. The run-in portion of this study evaluated the safety of weekly IV gedatolisib (PI3K/mTORi) and continuous daily talazoparib to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). A 3+3 design for dose escalation explored two dose levels. The phase II study began once the MTD and R2PD were confirmed. Eligibility required age ≥ 18, 1-2 prior lines of therapy (protocol later amended to allow up to 3 lines), and advanced TNBC or advanced HER2-negative BC with gBRCA1/2 mutation. The sample size for the phase II study was determined based on a 1-sided binomial test under the null and alternative ORR of 5% vs 20% with a type I error rate of 0.1 and power level of 80%. The primary endpoint was overall response rate (ORR) in TNBC without known gBRCA1/2 with secondary endpoints of progression-free survival (PFS) and overall survival (OS). Patients with a gBRCA1/2 mutation were not included in the primary endpoint analysis. Correlative studies are planned to evaluate HR deficiency mutations, PIK3CA mutations, and other exploratory genomics. A total of 33 patients were enrolled: 14 in the safety run-in phase of the trial and 19 in the phase II study (17 TNBC, 2 with gBRCA2 mutation). In the safety run-in, 6 patients were enrolled at dose level 1 (0.75 mg talazoparib po daily and 180 mg gedatolisib IV weekly) and 8 at dose level 2 (1 mg talazoparib po daily and 180 mg gedatolisib IV weekly). 42% of the cohort developed hyperglycemia, which was mostly grade 1. There was 1 DLT of grade 3 neutropenia at dose level 1. There were 3 patients who experienced grade 4 AEs, thrombocytopenia (2) and lymphopenia (1) which were outside of the DLT window. The MTD was 1 mg of talazoparib daily and 180 mg of gedatolisib weekly, and this was selected as the RP2D. A protocol amendment was made during the phase II portion to allow for a 3 week on/1 week off schedule for gedatolisib due to emerging data showing a more favorable safety profile and enhanced antitumor activity with this dosing schedule. In the 17 patients with TNBC and no gBRCA mutation in the phase II cohort, the ORR was 12%. Best response was partial response (PR) in 2 patients (12%), stable disease (SD) in 7 patients (41%), and progressive disease (PD) in 8 patients (47%). The clinical benefit rate (ORR+SD) at 16 weeks was 23.5%. The most common adverse events (AEs) in all 33 patients were anemia (70%), fatigue (67%), oral mucositis (64%), nausea (60%), neutropenia (45%) and anorexia (45%). Of these, most were grade 1-2 other than anemia (35% grade 3), neutropenia (20% grade 3), fatigue (18% grade 3), and oral mucositis (10% grade 3). There were no grade 4 AEs in the phase II study. Median PFS was approximately 3 months and median OS was approximately 6.4 months. Although this study did not meet its primary endpoint, there were 2 TNBC patients without a gBRCA1/2 mutation who achieved a partial response to this non-chemotherapy regimen. Future biomarker testing may help elucidate these findings and possible predictors of response. Citation Format: Sneha Phadke, Kari Wisinski, Oana Danciu, Ami Shah, Menggang Yu, Yi Chen, Kathy Miller, Mark Burkard. Phase 2 Trial with Safety Run-In of Gedatolisib Plus Talazoparib in Advanced Triple Negative or BRCA1/2 Positive, HER2 Negative Breast CancersBig Ten Cancer Research Consortium BTCRC-BRE18-337 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-09.

SIRT2 promotes base excision repair by transcriptionally activating OGG1 in an ATM/ATR-dependent manner.

Sirtuin 2 (SIRT2) regulates the maintenance of genome integrity by targeting pathways of DNA damage response and homologous recombination repair. However, whether and how SIRT2 promotes base excision repair (BER) remain to be determined. Here, we found that independent of its catalytic activity SIRT2 interacted with the critical glycosylase OGG1 to promote OGG1 recruitment to its own promoter upon oxidative stress, thereby enhancing OGG1 promoter activity and increasing BER efficiency. Further studies revealed that SIRT2 was phosphorylated on S46 and S53 by ATM/ATR upon oxidative stress, and SIRT2 phosphorylation enhanced the SIRT2-OGG1 interaction and mediated the stimulatory effect of SIRT2 on OGG1 promoter activity. We also characterized 37 cancer-derived SIRT2 mutants and found that 5 exhibited the loss of the stimulatory effects on OGG1 transcription. Together, our data reveal that SIRT2 acts as a tumor suppressor by promoting OGG1 transcription and increasing BER efficiency in an ATM/ATR-dependent manner.

Abstract 1987: PARP inhibitor (PARPi) rechallenge shows differential sensitivity in PARPi olaparib-resistant BRCA1-mutated (BRCA1m) high-grade serous ovarian cancer (HGSC)

Abstract Background: PARPis have changed the treatment paradigm in HGSC, particularly for women with BRCA mutations. PARPi, olaparib (O), is a standard of care maintenance therapy following platinum-based chemotherapy in newly diagnosed BRCA-mutated (BRCAm) HGSC. But patients ultimately develop resistance to O. We investigated whether rechallenge with different PARPi monotherapy could cause cytotoxicity in BRCAm HGSC after becoming O-resistant. We also aimed to identify possible mechanisms of actions of PARPi’s differential sensitivity. Methods: We developed a PARPi-resistant line (UWB-OlaJR) from BRCA1m PARPi-sensitive cells (UWB1.289) by gradient exposure of O with initial lower doses to a final concentration of 20 μM over 10 months. Four PARPis including O, niraparib (N), talazoparib (T), and veliparib (V) were tested. Cell growth was assessed using 5-day XTT and 10-day clonogenic assays. DNA damage and homologous recombination (HR) repair activity were evaluated using immunofluorescence (IF) staining of γH2AX and RAD51 foci, respectively. Replication fork (RF) dynamics was measured by DNA fiber assay. All data were repeated in triplicate and analyzed using t-test or one-way ANOVA. Data were shown as mean ± SD and P < 0.05 was considered statistically significant. Results: 5-day XTT assay shows a 53.7-fold increase in resistance to O in UWB-OlaJR relative to UWB1.289 (IC50 2.2 μM vs. 118.1 μM). Cross-resistance was observed with other 3 PARPis, showing increased IC50 values compared to its parental line (V: 23.4-fold, N: 160.5-fold, T: 229.6-fold). However, 10-day clonogenic assay showed no resistance against N (1.09-fold) and T (0.51-fold) in UWB-OlaJR, indicating differential sensitivity to different PARPis by a long-term cytotoxicity measure. Surprisingly, γH2AX foci did not increase in response to PARPis in UWB-OlaJR compared to UWB1.289 (O: 2.58-fold, V: 2.33-fold; N: 2.89-fold, T: 2.58-fold; all P < 0.05), suggesting possible restored DNA repair function. All four PARPis induced replication stress in both O-sensitive and -resistant cell lines as evidenced by decreased RF speed compared to untreated group (O: 0.57- and 0.7-fold, V: 0.68- and 0.74-fold, N: 0.47- and 0.47-fold, T: 0.47- and 0.52-fold; all P < 0.001). Notably, N and T treatments reduced fork speed to a greater extent than O and V in UWB-OlaJR. However, RF protection was not affected when UWB-OlaJR treated with different PARPis compared to UWB1.289 (O: 0.89-fold, V: 0.87-fold, N: 0.71-fold, T: 0.83-fold; all P < 0.05), suggesting that differential sensitivity to different PARPis was unlikely due to modulation of RF stabilization. Conclusions: Our findings demonstrate different PARPis may induce varied levels of cytotoxicity and replication stress in O-resistant BRCA1m HGSC, possibly via modulating alternative RF dynamics besides RF protection. Citation Format: Chi-Ting Shih, Tzu-Ting Huang, Jayakumar R. Nair, Jung-Min Lee. PARP inhibitor (PARPi) rechallenge shows differential sensitivity in PARPi olaparib-resistant BRCA1-mutated (BRCA1m) high-grade serous ovarian cancer (HGSC) [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 1987.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

A novel SIK2 inhibitor SIC-19 exhibits synthetic lethality with PARP inhibitors in ovarian cancer

PurposeOvarian cancer patients with HR proficiency (HRP) have had limited benefits from PARP inhibitor treatment, highlighting the need for improved therapeutic strategies. In this study, we developed a novel SIK2 inhibitor, SIC-19, and investigated its potential to enhance the sensitivity and expand the clinical utility of PARP inhibitors in ovarian cancer. MethodsThe SIK2 protein was modeled using a Molecular Operating Environment (MOE), and the most favorable model was selected based on a GBVI/WSA dG scoring function. The Chembridge Compound Library was screened, and the top 20 candidate compounds were tested for their interaction with SIK2 and downstream substrates, AKT-pS473 and MYLK-pS343. SIC-19 emerged as the most promising drug candidate and was further evaluated using multiple assays. ResultsSIC-19 exhibited selective and potent inhibition of SIK2, leading to its degradation through the ubiquitination pathway. The IC50 of SIC-19 correlated inversely with endogenous SIK2 expression in ovarian cancer cell lines. Treatment with SIC-19 significantly inhibited cancer cell growth and sensitized cells to PARP inhibitors in vitro, as well as in ovarian cancer organoids and xenograft models. Mechanistically, SIK2 knockdown and SIC-19 treatment reduced RAD50 phosphorylation at Ser635, prevented nuclear translocation of RAD50, disrupted nuclear filament assembly, and impaired DNA homologous recombination repair, ultimately inducing apoptosis. These findings highlight the crucial role of SIK2 in the DNA HR repair pathway and demonstrate the significant PARP inhibitor sensitization achieved by SIC-19 in ovarian cancer. ConclusionsSIC-19, a novel SIK2 inhibitor, effectively inhibits tumor cell growth in ovarian cancer by interfering with RAD50-mediated DNA HR repair. Furthermore, SIC-19 enhances the efficacy of PARP inhibitors, providing a promising therapeutic strategy to improve outcomes for ovarian cancer patients.

PPIP5K2 Facilitates Proliferation and Metastasis of Non-Small Lung Cancer (NSCLC) through AKT Signaling Pathway

Through facilitating DNA homologous recombination repair, PPIP5K2 has been proven to be essential for improving colorectal cancer survival in our previous research. However, its function in the tumorigenesis of NSCLC, the most common cancer and the primary cause of cancer-related death globally, is still unknown. Here, we initially discovered that PPIP5K2 had significant effects on proliferation of NSCLC cells through loss- and gain-of-function assays in vitro and in vivo. Moreover, PPIP5K2 is capable of regulating NSCLC cells metastasis in an EMT-dependent manner. In terms of mechanism exploration, we found that PPIP5K2 knockdown can significantly inhibit the phosphorylation of AKT/mTOR signaling pathway, whereas the overexpression of PPIP5K2 resulted in converse effects. By employing AKT signaling related agonists or antagonists, we further demonstrated that PPIP5K2 regulates NSCLC tumorigenesis partly via the AKT/mTOR pathway. In conclusion, PPIP5K2 plays a key oncogenic role in NSCLC by the activation of the AKT/mTOR signaling axis. It is anticipated that targeting PPIP5K2 might emerge as a viable therapeutic approach for NSCLC patients.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.