Recombinational Repair System Research Articles

Tricarboxylic Acid Cycle Relationships with Non-Metabolic Processes: A Short Story with DNA Repair and Its Consequences on Cancer Therapy Resistance.

Metabolic changes involving the tricarboxylic acid (TCA) cycle have been linked to different non-metabolic cell processes. Among them, apart from cancer and immunity, emerges the DNA damage response (DDR) and specifically DNA damage repair. The oncometabolites succinate, fumarate and 2-hydroxyglutarate (2HG) increase reactive oxygen species levels and create pseudohypoxia conditions that induce DNA damage and/or inhibit DNA repair. Additionally, by influencing DDR modulation, they establish direct relationships with DNA repair on at least four different pathways. The AlkB pathway deals with the removal of N-alkylation DNA and RNA damage that is inhibited by fumarate and 2HG. The MGMT pathway acts in the removal of O-alkylation DNA damage, and it is inhibited by the silencing of the MGMT gene promoter by 2HG and succinate. The other two pathways deal with the repair of double-strand breaks (DSBs) but with opposite effects: the FH pathway, which uses fumarate to help with the repair of this damage, and the chromatin remodeling pathway, in which oncometabolites inhibit its repair by impairing the homologous recombination repair (HRR) system. Since oncometabolites inhibit DNA repair, their removal from tumor cells will not always generate a positive response in cancer therapy. In fact, their presence contributes to longer survival and/or sensitization against tumor therapy in some cancer patients.

A phase IV trial to confirm the efficacy of olaparib in combination with bevacizumab as maintenance frontline treatment of HRD-positive ovarian tumours (IOlanTHe).

TPS5628 Background: PAOLA-1/ENGOT-ov25 trial demonstrated that Olaparib (O) plus Bevacizumab (B) determined a significant survival benefit in patients (pts) with advanced, high-grade, epithelial ovarian, peritoneal and/or fallopian-tube cancer (HEOC) presenting defects of homologous recombination repair system (HRD+) in clinical response after first-line platinum-based chemotherapy (CT) plus B. Methods: IOLANTHE is a multicenter Italian phase IV trial (sponsored by the MaNGO group, Protocol Number IRFMN-OVA-8542) aiming to confirm in a setting close to clinical practice the efficacy of this combination in pts with HRD+HEOC. As the study includes a consistent translational component, all suspicious advanced cases will be considered for study enrollment. Pts with a confirmed pathological diagnosis of HEOC will be included in the step 1 of the trial which main inclusion criteria are the following: -availability of formalin-fixed, paraffin-embedded samples for the central testing of HRD status (performed by Myriad Mychoice CDx Plus assay) - suitability to receive CT + B. Only HRD+ pts responding to first line CT plus at least one cycle of B will be enrolled in the step 2 of the trial and will be treated with B 15 mg/kg every 3 weeks plus O 300 mg twice daily. Considering the PAOLA-1 median progression free survival (PFS) of 37.2 months (mo) in the B-O arm, with a sample size of 90 pts followed for 24 months and setting a one-side error of 5%, we will have a statistical power of 80% to demonstrate a PFS-24mo≥64% with an upper critical value of 61%. In order to have 90 patients eligible for the step 2 of the study, 190 pts are needed to be enrolled in the step 1 of the clinical study (as approximately 80% of pts with HEOC will respond to CT and out of these, 60% will be HRD+). The translational subproject 1 will consist of the analysis of circulating-tumor (ctDNA), using whole genome sequencing aiming - to correlate residual tumour after surgery and ctDNA levels - to anticipate the diagnosis of progression - to monitor the mutational status of HR-related genes and other genes such as Tp53BP1, POLQ, REV7 probably involved in PARPi resistance during the maintenance therapy. The translational subproject 2 will be developed to compare pts’ response to therapy with that of cancer cells, tested by organotypic model treated with the combination. These models will be generated using fresh tumor tissue and ascitic fluid for the isolation of the tumor cells and macroscopically healthy omentum for the isolation of the mesothelial cells and fibroblasts. Eight centers have been activated and other 6 are waiting for the activation with 19 patients registered and 3 screening failures. The enrollment is continuing, according to protocol timeline (12 months for enrollment, 6 months for surgery and CT and 24 months for maintenance treatment). Clinical trial information: NCT06121401 .

A molecular glue RBM39-degrader induces synthetic lethality in cancer cells with homologous recombination repair deficiency

E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.

Classical and novel properties of Holliday junction resolvase SynRuvC from Synechocystis sp. PCC6803.

Cyanobacteria, which have a photoautotrophic lifestyle, are threatened by ultraviolet solar rays and the reactive oxygen species generated during photosynthesis. They can adapt to environmental conditions primarily because of their DNA damage response and repair mechanisms, notably an efficient homologous recombination repair system. However, research on double-strand break (DSB) repair pathways, including the Holliday junction (HJ) resolution process, in Synechocystis sp. PCC6803 is limited. Here, we report that SynRuvC from cyanobacteria Synechocystis sp. PCC6803 has classical HJ resolution activity. We investigated the structural specificity, sequence preference, and biochemical properties of SynRuvC. SynRuvC strongly preferred Mn2+ as a cofactor, and its cleavage site predominantly resides within the 5'-TG↓(G/A)-3' sequence. Interestingly, novel flap endonuclease and replication fork intermediate cleavage activities of SynRuvC were also determined, which distinguish it from other reported RuvCs. To explore the effect of SynRuvC on cell viability, we constructed a knockdown mutant and an overexpression strain of Synechocystis sp. PCC6803 (synruvCKD and synruvCOE) and assessed their survival under a variety of conditions. Knockdown of synruvC increased the sensitivity of cells to MMS, HU, and H2O2. The findings suggest that a novel RuvC family HJ resolvase SynRuvC is important in a variety of DNA repair processes and stress resistance in Synechocystis sp. PCC6803.

Homologous Recombination Deficiency (HRD) Scoring, by Means of Two Different Shallow Whole-Genome Sequencing Pipelines (sWGS), in Ovarian Cancer Patients: A Comparison with Myriad MyChoice Assay.

High-grade serous ovarian cancer (HGSOC) patients carrying the BRCA1/2 mutation or deficient in the homologous recombination repair system (HRD) generally benefit from treatment with PARP inhibitors. Some international recommendations suggest that BRCA1/2 genetic testing should be offered for all newly diagnosed epithelial ovarian cancer, along with HRD assessment. Academic tests (ATs) are continuously under development, in order to break down the barriers patients encounter in accessing HRD testing. Two different methods for shallow whole-genome sequencing (sWGS) were compared to the reference assay, Myriad. All these three assays were performed on 20 retrospective HGSOC samples. Moreover, HRD results were correlated with the progression-free survival rate (PFS). Both sWGS chemistries showed good correlation with each other and a complete agreement, even when compared to the Myriad score. Our academic HRD assay categorized patients as HRD-Deficient, HRM-Mild and HRN-Negative. These three groups were matched with PFS, providing interesting findings in terms of HRD scoring and months of survival. Both our sWGS assays and the Myriad test correlated with the patient's response to treatments. Finally, our AT confirms its capability of determining HRD status, with the advantage of being faster, cheaper, and easier to carry out. Our results showed a prognostic value for the HRD score.

A BRCA2 germline mutation and high expression of immune checkpoints in a TNBC patient

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.

An Overview of PARP Resistance in Ovarian Cancer from a Molecular and Clinical Perspective.

Epithelial ovarian cancer (EOC), a primarily high-grade serous carcinoma (HGSOC), is one of the major causes of high death-to-incidence ratios of all gynecological cancers. Cytoreductive surgery and platinum-based chemotherapy represent the main treatments for this aggressive disease. Molecular characterization of HGSOC has revealed that up to 50% of cases have a deficiency in the homologous recombination repair (HRR) system, which makes these tumors sensitive to poly ADP-ribose inhibitors (PARP-is). However, drug resistance often occurs and overcoming it represents a big challenge. A number of strategies are under investigation, with the most promising being combinations of PARP-is with antiangiogenetic agents and immune checkpoint inhibitors. Moreover, new drugs targeting different pathways, including the ATR-CHK1-WEE1, the PI3K-AKT and the RAS/RAF/MEK, are under development both in phase I and II-III clinical trials. Nevertheless, there is still a long way to go, and the next few years promise to be exciting.

Exploring the prognostic and predictive impact of genomic loss of heterozygosity (LOH) in patients with metastatic colorectal cancer (mCRC).

3576 Background: Genomic LOH consists in the loss of chromosomal regions and is associated with the homologous recombination repair (HRR) system deficiency (dHRR). In ovarian cancer, LOH-high predicts benefit from platinum-based chemotherapy and PARP inhibitors. In mCRC, the role of LOH has been poorly investigated. Methods: An NGS-based assay (FoundationOne CDx; Foundation Medicine, Inc. Cambrige, MA) was used to determine the percentage of genomic LOH and the presence of pathogenetic mutations in HRR-related genes in archival chemo-naïve tumor tissues of mCRC patients included in a real-world registry and in the AtezoTRIBE (NCT03721653) and AVETRIC (NCT04513951) studies. Both these trials assessed the combination of an anti-PDL1 (atezolizumab or avelumab) with the triplet FOLFOXIRI plus bevacizumab or cetuximab. The prespecified cut-off of ≥14.08 for LOH-high described for mCRC (Sokol et al., JCO Precis Oncol 2020) was adopted. Tumors with at least one biallelic alteration in any of the 27 genes involved in the HRR pathway (Riaz et al., Nat Commun 2017) included in the FoundationOne CDx panel (BARD1, BRCA1, BRCA2, BRIP1, MRE11A, NBN, PALB2, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RBBP8, XRCC2, ABL1, ATM, ATR, BAP1, CDK12, DNMT3A, ERCC4, FANCA, FANCC, FANCG, FANCL, PARP1) were defined dHRR. Results: Overall, 196 samples were analysed. None of 7 MSI-H tumors were classified as LOH-high. Fourteen (7%) and 6 (3%) of 189 MSS tumors were classified as LOH-high and dHRR, respectively. In particular, in LOH-high subgroup, 3 (21%) tumors were defined dHRR, while 3 (50%) tumors were classified LOH-high among dHRR tumors. LOH-high tumors were more frequently BRAF mutated (p = 0.019) and dHRR (p = 0.006) compared to LOH-low. Among patients receiving triplet chemotherapy +/- biologic agent alone (N = 55) or with an anti-PDL1 (N = 58), an interaction effect was shown between the effect of the addition of the checkpoint inhibitor and LOH with higher benefit in the LOH-high subgroup (N = 10) in terms of both PFS (pinteraction= 0.002) and OS (pinteraction< 0.001). In the cohort of patients not receiving the anti-PDL1, longer PFS was observed in patients with LOH-low (N = 125) respect to LOH-high (N = 6) tumors (12.1 vs 5.1 months, HR: 0.11, 95%CI: 0.04-0.26, p < 0.001). No differences in PFS or OS were reported between patients treated with first-line oxaliplatin- (N = 40) vs irinotecan-based doublets (N = 25) or with the triplet FOLFOXIRI (N = 55) vs doublets (N = 65) according to LOH status. No prognostic or predictive impact of HRR deficiency was shown. Conclusions: In MSS mCRC, LOH-high was associated with biallelic alterations in the HRR system, worse prognosis and higher benefit from the addition of anti-PDL1 agents to chemotherapy. Considering the low number of LOH-high tumors in our study, these results deserve confirmation in larger cohorts.

Overlapping gene dependencies for PARP inhibitors and carboplatin response identified by functional CRISPR-Cas9 screening in ovarian cancer

PARP inhibitors (PARPi) have revolutionized the therapeutic landscape of epithelial ovarian cancer (EOC) treatment with outstanding benefits in regard to progression-free survival, especially in patients either carrying BRCA1/2 mutations or harboring defects in the homologous recombination repair system. Yet, it remains uncertain which PARPi to apply and how to predict responders when platinum sensitivity is unknown. To shed light on the predictive power of genes previously suggested to be associated with PARPi response, we systematically reviewed the literature and identified 79 publications investigating a total of 93 genes. The top candidate genes were further tested using a comprehensive CRISPR-Cas9 mutagenesis screening in combination with olaparib treatment. Therefore, we generated six constitutive Cas9+ EOC cell lines and profiled 33 genes in a CRISPR-Cas9 cell competition assay using non-essential (AAVS1) and essential (RPA3 and PCNA) genes for cell fitness as negative and positive controls, respectively. We identified only ATM, MUS81, NBN, BRCA2, and RAD51B as predictive markers for olaparib response. As the major survival benefit of PARPi treatment was reported in platinum-sensitive tumors, we next assessed nine top candidate genes in combination with three PARPi and carboplatin. Interestingly, we observed similar dropout rates in a gene and compound independent manner, supporting the strong correlation of cancer cell response to compounds that rely on DNA repair for their effectiveness. In addition, we report on CDK12 as a common vulnerability for EOC cell survival and proliferation without altering the olaparib response, highlighting its potential as a therapeutic target in EOC.

Biochemical and Structural Study of RuvC and YqgF from Deinococcus radiodurans.

ABSTRACTDeinococcus radiodurans possesses robust DNA damage response and repair abilities, and this is mainly due to its efficient homologous recombination repair system, which incorporates an uncharacterized Holliday junction (HJ) resolution process. D. radiodurans encodes two putative HJ resolvase (HJR) homologs: RuvC (DrRuvC) and YqgF (DrYqgF). Here, both DrRuvC and DrYqgF were identified as essential proteins for the survival of D. radiodurans. The crystal structures and the biochemical properties of DrRuvC and DrYqgF were also studied. DrRuvC crystallized as a homodimer, while DrYqgF crystallized as a monomer. DrRuvC could preferentially cleave HJ at the consensus 5′-(G/C)TC↓(G/C)-3′ sequence and could prefer using Mn2+ for catalysis in vitro, which would be different from the preferences of the other previously characterized RuvCs. On the other hand, DrYqgF was identified as a Mn2+-dependent RNA 5′–3′ exo/endonuclease with a sequence preference for poly(A) and without any HJR activity.

Tumor Genotyping and Homologous Recombination Repair Gene Variants in Patients With Epithelial Ovarian Cancer: Is Pathogenic Enough?

Our hypothesis was that the predictive accuracy of pathogenic variants in genes participating in the homologous recombination repair (HRR) system in patients with epithelial ovarian cancer (EOC) could be improved by considering additional next-generation sequencing (NGS) metrics. NGS genotyping was performed in tumor tissue, retrospectively and prospectively collected from patients with EOC, diagnosed from 8/1998 to 10/2016. Variants were considered clonal when variant allele frequencies corresponded to >25%. The primary endpoint was overall survival (OS). This study included 501 patients with EOC, predominantly with high-grade serous (75.2%) and advanced stage tumors (81.7%); median age was 58 years (22-84). Pathogenic and clonal pathogenic variants in HRR and/or TP53 genes were identified in 72.8% and 66.5% tumors, respectively. With a median follow-up of 123.9 months, the presence of either pathogenic or clonal pathogenic HRR-only variants was associated with longer OS compared to HRR/TP53 co-mutation (HR=0.54; 95% CI, 0.34-0.87, Wald’s p=0.012 and HR=0.45; 95% CI, 0.27-0.78, Wald’s p=0.004, respectively). However, only the presence of clonal HRR-only variants was independently associated with improved OS (HR=0.55; 95% CI, 0.32-0.94, p=0.030). Variant clonality and co-occuring TP53 variants affect the predictive value of HRR pathogenic variants for platinum agents in patients with EOC.Clinical Trial Registration[ClinicalTrials.gov], identifier [NCT04716374].

Inhibition of DNA Repair in Combination with Temozolomide or Dianhydrogalactiol Overcomes Temozolomide-Resistant Glioma Cells.

Simple SummaryGlioblastoma is the most prevalent and lethal brain tumor. Temozolomide is usually used for the treatment of glioblastoma. The poor prognosis of the tumor is due to drug resistance and tumor heterogeneity. The mechanism of the resistance to temozolomide is various within the same tumor. The aim of the study was to clarify the mechanism of temozolomide resistance and find methods to overcome temozolomide resistance in glioma. Inhibition of DNA repair (homologous recombination or base excision repair) resensitized resistant cells harboring different resistance mechanism to temozolomide. Additionally, a bifunctional DNA-targeting agent, dianhydrogalactiol, showed anti-tumor effect independent of MGMT and mismatch repair status. Further, inhibition of checkpoint or homologous recombination enhanced dianhydrogalactiol-induced cytotoxicity in temozolomide-resistant glioma cells. Although resistance to temozolomide is clinically important issue, selecting suitable treatments for resistance mechanism can improve the prognosis of glioma.Resistance to temozolomide and intratumoral heterogeneity contribute to the poor prognosis of glioma. The mechanisms of temozolomide resistance can vary within a heterogeneous tumor. Temozolomide adds a methyl group to DNA. The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to a futile DNA mismatch repair cycle, formation of double-strand breaks, and eventual cell death when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair (BER). The study aim was to elucidate temozolomide resistance mechanisms and identify methods to overcome temozolomide resistance in glioma. Several temozolomide-resistant clones were analyzed. Increased homologous recombination and mismatch repair system deficiencies contributed to temozolomide resistance. Inhibition of homologous recombination resensitized resistant cells with high homologous recombination efficiency. For the mismatch repair-deficient cells, inhibition of BER by PARP inhibitor potentiated temozolomide-induced cytotoxicity. Dianhydrogalactiol is a bifunctional DNA-targeting agent that forms N7-alkylguanine and inter-strand DNA crosslinks. Dianhydrogalactiol reduced the proliferation of cells independent of MGMT and mismatch repair, inducing DNA double-strand breaks and apoptosis in temozolomide-resistant cells. Further, inhibition of chk1 or homologous recombination enhanced dianhydrogalactiol-induced cytotoxicity in the cells. Selecting treatments most appropriate to the types of resistance mechanisms can potentially improve the prognosis of glioma.

Abstract 2711: Mechanism of natural progression from low grade glioma to glioblastoma: A case report

Abstract Introduction: Current studies about the progression mechanisms of gliomas mainly focus on the progression under treatment selection. Little is known about how low grade gliomas (LGG) progress to glioblastoma (GBM) under natural pressure, which remains to be elucidated. Methods: We reported a 67-years-old woman, whose intracranial lesion was diagnosed first in 2010 but without treatment. An enhanced peripheral lesion was detected around the primary lesion in 2018. Both the primary and secondary lesions were resected and diagnosed as diffuse astrocytoma(LGG) and glioblastoma (GBM), respectively. Both samples were subjected to whole exome sequencing, RNA sequencing, methylation array and TCR sequencing. Results: The results showed that both samples were IDH1 mutant, 1p/19q non-codel, TERT promoter wild-type, G-CIMP High and MGMT methylated. About 50% of the mutant genes were identical in both samples, suggesting that both samples had the same origin. Comparing with the primary glioma (LGG), the mutant allele fractions (MAFs) of these shared genes were increased in the secondary lesion (GBM). Furthermore, the MAFs of a novel mutation (p.N219K) in the gene NAF1 increased from 44% to 75%, and the ratio of the corresponding RNA reads increased from 37.5% to 75%, which indicating NAF1(p.N219K) maybe a novel driver mutation promoting tumor progression in LGG. The unique genes in the secondary lesion (GBM) with MAFs>15% were CIC, HMCN1, RPA4 and BRAC2. As it is well known, RPA4 and BRCA2 are involved in homologous recombination repair system. The ssGSEA analysis revealed that KRAS pathway and NF-kB pathway was activated in the secondary lesion (GBM). Mast cells were increased in the GBM, whereas CD4+ memory T cells and CD8+ T cells decreased, which suggesting potentiation of innate immune response and suppression of adaptive immune response. Surprisingly, although only 2.13% (n=14) of TCR sequences detected in the blood were shared in the LGG or the GBM, their reads accounted for 44% of all TCR sequences reads in blood, 71.10% in the LGG and 46.40% in the GBM, suggesting these T cells may play vital functions in the process of glioma progression. Conclusion: Here, we reported an IDHmut-1p/19q-noncodel glioma case with natural evolution. Several biological processes were identified in this progression including deficiency of DNA damage repair, activation of KRAS pathway and NF-kB pathway, excitation of innate immune response and suppression of adaptive immune response. We uncovered a novel driver mutation (NAF1, p.N219K), further analyses of its role in the natural progression of glioma are expected. We also found several significantly proliferated T cell clones in the development of gliomas, which suggesting the promise of T cells in clinical diagnosis and treatment of glioma. Citation Format: Long Wang, Xuegang Li, Tunan Chen, Feilong Zhao, Tonghui Ma, Hua Feng, Fei Li. Mechanism of natural progression from low grade glioma to glioblastoma: A case report [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2711.

Combinatorial Therapy of High Dose Vitamin C and PARP Inhibitors in DNA Repair Deficiency: A Series of 8 Patients.

Background:Tumor-specific DNA repair defects are ubiquitous in cancerous tissue, which offers a potential for clinical gain to build on these perturbations. Intravenous high-dose vitamin C (IVC) triggers the formation of hydrogen peroxide (H2O2), which contributes to Fenton chemistry producing hydroxyl radicals (-OH), causing selective damage to DNA. Herein, we evaluated the therapeutic response to IVC and PARP inhibitors (PARPi) in combination in 8 patients with a deficiency of homologous recombination repair system (dHRR) in a 3-year period.Material and Methods:Eight patients with progressive stage IV malignancy, who were pre-treated with conventional methods, were admitted to our clinic. Subsequent therapy has included IVC at a dose that was set to be in the range of 1 to 1.5 g/kg, although 1.25 g/kg was dominantly administered. Furthermore, following genomic evaluation, PARPi (niraparib or olaparib or talazoparib) was chosen to be used in combination with IVC which was administered 2 to 4 times a week.Results:In the present study, we achieved partial response in 5 patients and complete response in 3 patients. Grade 2 anemia and fatigue toxicities were observed in some cases, while grade 3 toxicity was not found in any of the patients.Conclusion:Our 8 patient case study shows that IVC could be a plausible additional therapy for HRR deficiency. Although the single agent PARPi therapy is effective for metastatic disease, an overall survival (OS) advantage has not been demonstrated. Our results suggest that adding IVC can improve PARPi therapy outcomes. To fully endorse the results stated above, combinatorial therapy of intravenous high dose vitamin C and PARP inhibitors needs to be reviewed in broader cohorts of patients.

CRISPR-Cas9-mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes.

Prkdc encodes for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) playing a key role in nonhomologous end joining pathway during DNA double-strand break repair and also influencing the homologous recombination (HR) repair system by phosphorylation of proteins involved in HR. In addition, Prkdc has other critical functions in biological processes, such as transcriptional regulation, telomere stability, apoptosis, and metabolism. DNA-PKcs upregulates during in vitro differentiation of mouse embryonic stem cells (mESCs). To address the potential role of Prkdc in mESCs pluripotency and in vitro differentiation into ectoderm, mesoderm, and endoderm germ layers under normal physiological conditions, a bi-allelic Prkdc-knockout cell line was generated in the present study by employing CRISPR/Cas9 system, and subsequently, its potential role in stemness and development was studied. The results of the study showed that the expression of pluripotency-associated genes, including Nanog and Sox-2 were overexpressed in the bi-allelic Prkdc-knockout cell line. Also, bi-allelic Prkdc-knockout cell line was shown to have typical mESCs cell morphology, cell cycle distribution, and alkaline phosphatase activity. Furthermore, the results of the study revealed that the expression of several germ layer markers is modulated in Prkdc-knockout lines. In conclusion, the findings of our study demonstrated the role of Prkdc during differentiation and development of ESCs.

Phosphodiesterase Type 5 (PDE5) Inhibitors Sensitize Topoisomerase II Inhibitors in Killing Prostate Cancer Through PDE5-Independent Impairment of HR and NHEJ DNA Repair Systems.

Human castration-resistant prostate cancer (CRPC) is a significant target of clinical research. The use of DNA-damaging agents has a long history in cancer chemotherapy but is limited by their toxicities. The combination with a safer drug can be a strategy in reducing dosage and toxicity while increasing anticancer activity in CRPC treatment. Phosphodiesterase type 5 (PDE5) inhibitors are used to treat erectile dysfunction through the selective inhibition of PDE5 that is responsible for cGMP degradation in the corpus cavernosum. Several studies have reported that PDE5 inhibitors display protective effect against doxorubicin-induced cardiotoxicity. The combinatory treatment of CRPC with doxorubicin and PDE5 inhibitors has been studied accordingly. The data demonstrated that sildenafil or vardenafil (two structure-related PDE5 inhibitors) but not tadalafil (structure-unrelated to sildenafil) sensitized doxorubicin-induced apoptosis in CRPC cells with deteriorating the down-regulation of anti-apoptotic Bcl-2 family members, including Bcl-xL and Mcl-1, and amplifying caspase activation. Homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair systems were inhibited in the apoptotic sensitization through detection of nuclear foci formation of Rad51 and DNA end-binding of Ku80. PDE5 knockdown to mimic the exposure to PDE5 inhibitors did not reproduce apoptotic sensitization, suggesting a PDE5-independent mechanism. Not only doxorubicin, sildenafil combined with other inhibitors of topoisomerase II but not topoisomerase I also triggered apoptotic sensitization. In conclusion, the data suggest that sildenafil and vardenafil induce PDE5-independent apoptotic sensitization to doxorubicin (or other topoisomerase II inhibitors) through impairment of both HR and NHEJ repair systems that are evident by a decrease of nuclear Rad51 levels and their foci formation in the nucleus, and an inhibition of Ku80 DNA end-binding capability. The combinatory treatment may enable an important strategy for anti-CRPC development.

A new role for Drosophila Aurora-A in maintaining chromosome integrity.

Aurora-A is a conserved mitotic kinase overexpressed in many types of cancer. Growing evidence shows that Aurora-A plays a crucial role in DNA damage response (DDR) although this aspect has been less characterized. We isolated a new aur-A mutation, named aur-A949, in Drosophila, and we showed that it causes chromosome aberrations (CABs). In addition, aur-A949 mutants were sensitive to X-ray treatment and showed impaired γ-H2Av foci dissolution kinetics. To identify the pathway in which Aur-A works, we conducted an epistasis analysis by evaluating CAB frequencies in double mutants carrying aur-A949 mutation combined to mutations in genes related to DNA damage response (DDR). We found that mutations in tefu (ATM) and in the histone variant H2Av were epistatic over aur-A949 indicating that Aur-A works in DDR and that it is required for γ-H2Av foci dissolution. More interestingly, we found that a mutation in lig4, a gene belonging to the non-homologous end joining (NHEJ) repair pathway, was epistatic over aur-A949. Based on studies in other systems, which show that phosphorylation is important to target Lig4 for degradation, we hypothesized that in aur-A949 mutant cells, there is a persistence of Lig4 that could be, in the end, responsible for CABs. Finally, we observed a synergistic interaction between Aur-A and the homologous recombination (HR) repair system component Rad 51 in the process that converts chromatid deletions into isochromatid deletions. Altogether, these data indicate that Aur-A depletion can elicit chromosome damage. This conclusion should be taken into consideration, since some anticancer therapies are aimed at reducing Aurora-A expression.

BRCA Status Does Not Predict Synergism of a Carboplatin and Olaparib Combination in High-Grade Serous Ovarian Cancer Cell Lines.

Over 50% of epithelial ovarian cancers express the BRCAness profile that leads to a dysfunctional homologous recombination repair system. The combination of a dysfunctional homologous recombination repair system and a poly(ADP-ribose) polymerase (PARP) inhibitor results in a synthetic lethal phenotype. The PARP inhibitor olaparib, approved as a monotherapy for patients with a germline BRCA mutation, has shown promising results in preclinical studies when combined with DNA damaging agents, such as carboplatin. However, dose-limiting toxicities have hindered the use of a combination therapy with olaparib in the clinical setting. By concurrent administration of carboplatin and olaparib at various molar ratios of drugs, the aim of this study was to explore the optimal dosing ratio of carboplatin-olaparib combinations in a comprehensive panel of eight BRCA-proficient and -deficient high-grade serous ovarian cancer (HGSOC) cell lines. Overall, synergy was observed in the BRCA1/2-mutated or defective cell lines when olaparib was combined at lower molar ratios of olaparib to carboplatin. Immunostaining of γH2AX foci revealed increased DNA damage as a result of this synergistic drug combination in the UWB1.289 paired cell lines. In vitro activity of the individual agents, carboplatin and olaparib, did not correlate with PARP1 expression in each cell line. Importantly, synergism was also observed in a subset of BRCA wild-type cell lines (OV90 and PEO4) suggesting therapeutic benefits of this combination beyond BRCA-dependent synthetic lethality. The administration of drugs at synergistic ratios has the potential to increase efficacy and reduce toxicity.

Polymorphisms in homologous recombination repair genes and the risk and survival of breast cancer.

Immunoglobulin (Ig)A antibody of Epstein-Barr virus (EBV) was found to associate with breast cancer (BC), whereas IgA positivity was related to a series of genetic markers in the genes of homologous recombination repair system (HRRs). We assessed the associations of the polymorphisms in HRR genes with the risk and survival of BC. A case-control study was conducted with 1551bc cases and 1605 age-matched healthy controls between October 2008 and March 2012 in the Guangzhou Breast Cancer Study (GZBCS), China, and the case population were followed up until 31 January 2016. Five single nucleotide polymorphisms of candidate genes in HRR system were genotyped. Odds ratios (ORs) and hazards ratios (HRs) were calculated using multivariate logistic regression and Cox proportional hazards regression to estimate the risk and prognostic effect, respectively. RFC1 rs6829064 (AA) was associated with an increased BC risk [OR=1.35; 95% confidence interval (CI)=1.06-1.73] compared to the wild genotype (GG). NRM rs1075496 (GT/TT versus GG) was associated with a worse progression-free survival (PFS) and the HR was 1.34 (95% CI=1.01-1.78), particularly among advanced patients. LIG3 rs1052536 (CT/TT versus CC) was associated with a better PFS and the HR was 0.70 (95% CI=0.53-0.93). However, RAD54L rs1710286 and RPA1 rs11078676 were not observed to be associated with either the risk or survival of BC. The findings of the present study suggest that the polymorphisms in HRR genes were associated with BC risk (RFC1 rs6829064) and prognosis (NRM rs1075496 and LIG3 rs1052536), whereas RAD54L rs1710286 and RPA1 rs11078676 had null associations with BC.

Screening of Pesticides with the Potential of Inducing DSB and Successive Recombinational Repair.

A study was realized to ascertain whether eight selected pesticides would induce double strand breaks (DSB) in lymphocyte cultures and whether this damage would induce greater levels of proteins Rad51 participating in homologous recombination or of p-Ku80 participating in nonhomologous end joining. Only five pesticides were found to induce DSB of which only glyphosate and paraoxon induced a significant increase of p-Ku80 protein, indicating that nonhomologous end joining recombinational DNA repair system would be activated. The type of gamma-H2AX foci observed was comparable to that induced by etoposide at similar concentrations. These results are of importance since these effects occurred at low concentrations in the micromolar range, in acute treatments to the cells. Effects over longer exposures in actual environmental settings are expected to produce cumulative damage if repeated events of recombination take place over time.