Homologous Recombination DNA Research Articles

Predictive molecular genetic tests in clinical oncology

Molecular genetic testing is an essential component of modern practical oncology. It is aimed at individualized therapy prescription and can significantly improve the life expectancy of patients. The analyzed molecular events include alterations affecting single genes (EGFR, KRAS, etc.), as well as integral characteristics of tumor genome, for example, total tumor mutation burden (TMB) or deficiency of certain types of DNA repair (microsatellite instability, homologous DNA recombination deficiency, etc.). The spectrum of clinically relevant genetic markers continues to expand, and some of them acquire the status of "agnostic", i.e., applicable to tumors of various origin. As a consequence, there is a trend toward substitution of individual gene testing with the use of multigene panels allowing simultaneous assessment of all significant predictive markers.

Clinical Roles of Risk Model Based on Differentially Expressed Genes in Mesenchymal Stem Cells in Prognosis and Immunity of Non-small Cell Lung Cancer.

The tumor microenvironment (TME) plays an important regulatory role in the progression of non-small cell lung cancer (NSCLC). Mesenchymal stem cells (MSCs) in the TME might contribute to the occurrence and development of cancer. This study evaluates the role of differentially expressed genes (DEGs) of MSCs and the development of NSCLC and develops a prognostic risk model to assess the therapeutic responses. The DEGs in MSCs from lung tissues and from normal tissues were analyzed using GEO2R. The functions and mechanisms of the DEGs were analyzed using the Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Additionally, the Cancer Genome Atlas (TCGA) database was used to determine the expression levels of the DEGs of MSCs in the NSCLC tissues. The prognostic factors of NSCLC related to MSCs were screened by survival analysis, meta-analysis, Cox regression analysis, and a prognostic risk model and nomogram was developed. The signaling mechanisms and immune roles that risk model participate in NSCLC development were determined via Gene Set Enrichment Analysis and CIBERSORT analysis. Compared to the normal tissues, 161 DEGs were identified in the MSCs of the lung tissues. These DEGs were associated with mechanisms, such as DNA replication, nuclear division, and homologous recombination. The overexpression of DDIT4, IL6, ITGA11, MME, MSX2, POSTN, and TRPA1 were associated with dismal prognosis of NSCLC patients. A high-risk score based on the prognostic risk model indicated the dismal prognosis of NSCLC patients. The nomogram showed that the age, clinical stage, and risk score affected the prognosis of NSCLC patients. Further, the high-risk model was associated with signaling mechanisms, such as the ECM-receptor interaction pathways, cytokine-cytokine receptor interaction, and MAPK pathways, involved in the progression of NSCLC and was also related to the components of the immune system, such as macrophages M0, T follicular helper cells, regulatory T cells. Therefore, the risk model and nomogram that was constructed on the basis of MSC-related factors such as POSTN, TRPA1, and DDIT4 could facilitate the discovery of target molecules that participate in the progression of NSCLC, which might also serve as new candidate markers for evaluating the prognosis of NSCLC patients.

Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer

The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generate spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes. We identify a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we report a prognostic role of a proliferative tumor-cell subpopulation, which associates with enhanced spatial tumor-immune interactions by CD8+ and CD4 + T-cells in the BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the potential to improve immunotherapeutic strategies and patient stratification in HGSC.

CDK5RAP3, a New BRCA2 Partner That Regulates DNA Repair, Is Associated with Breast Cancer Survival.

Simple SummaryBRCA2 is an essential gene for DNA repair by homologous recombination and is often mutated in families at risk of breast and ovarian cancer. In this study we identified CDK5RAP3 tumor suppressor as a new BRCA2-interacting protein. CDK5RAP3 negatively regulates DNA repair of double-strand breaks, providing a new mechanism of DNA damage resistance. Consistently, gene expression data analysis showed CDK5RAP3 overexpression in breast cancer is associated with poorer survival. Finally, we identified common genetic variations in the CDK5RAP3 locus as potentially associated with breast and ovarian cancer risk in a large cohort of BRCA1 and BRCA2 mutation carriers.BRCA2 is essential for homologous recombination DNA repair. BRCA2 mutations lead to genome instability and increased risk of breast and ovarian cancer. Similarly, mutations in BRCA2-interacting proteins are also known to modulate sensitivity to DNA damage agents and are established cancer risk factors. Here we identify the tumor suppressor CDK5RAP3 as a novel BRCA2 helical domain-interacting protein. CDK5RAP3 depletion induced DNA damage resistance, homologous recombination and single-strand annealing upregulation, and reduced spontaneous and DNA damage-induced genomic instability, suggesting that CDK5RAP3 negatively regulates double-strand break repair in the S-phase. Consistent with this cellular phenotype, analysis of transcriptomic data revealed an association between low CDK5RAP3 tumor expression and poor survival of breast cancer patients. Finally, we identified common genetic variations in the CDK5RAP3 locus as potentially associated with breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. Our results uncover CDK5RAP3 as a critical player in DNA repair and breast cancer outcomes.

BRD4 inhibition induces synthetic lethality in ARID2-deficient hepatocellular carcinoma by increasing DNA damage.

Hepatocellular carcinoma (HCC) has emerged as the third cause of cancer-related death owing to lacking effective systemic therapies. Genomic DNA sequencing revealed the high frequency of loss-of-function mutations in ARID2, which encodes a subunit of SWI/SNF chromatin remodeling complex, however, the therapeutic strategy for the HCC patients with ARID2 mutations is still completely unclear. In this study, we first performed a high-throughput screening approach using a compound library consisting of 2 180 FDA-approved drugs and other compounds, to elicit the potential drugs for synthetic lethality to target ARID2-deficient HCC cells. Interestingly, JQ1, a selective inhibitor of bromodomain protein BRD4, uniquely suppressed the growth of ARID2- deficient HCC cells. Next JQ1 is further confirmed to predominantly induce cell lethality upon ARID2 depletion through exacerbating DNA damage, especially double strand breaks (DSBs). Functional assays demonstrated that both BRD4 inhibition and ARID2 deficiency synergistically impede two main DNA damage repair pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ), through attenuating the transcription of BRCA1, RAD51, and 53BP1, which encode the core molecules responsible for DSB repair. Mechanistically, both ARID2 and BRD4 exert a synergistic effect for maintaining transcriptional enhancer-promoter loops of these genes within chromatin conformation. However, as both ARID2 and BRD4 are disrupted, the expression of these DNA repair-related genes in response to DNA damage are hindered, resulting in DSB accumulation and cell apoptosis. Taken together, this study discloses that BRD4 inhibition may induce synthetic lethality in ARID2-deficient HCC cells, which might provide a potential therapeutic strategy for HCC patients with ARID2 mutations.

Alteration in glycolytic/cholesterogenic gene expression is associated with bladder cancer prognosis and immune cell infiltration

BackgroundOncogenic metabolic reprogramming contributes to tumor growth and immune evasion. The intertumoral metabolic heterogeneity and interaction of distinct metabolic pathways may determine patient outcomes. In this study, we aim to determine the clinical and immunological significance of metabolic subtypes according to the expression levels of genes related to glycolysis and cholesterol-synthesis in bladder cancer (BCa).MethodsBased on the median expression levels of glycolytic and cholesterogenic genes, patients were stratified into 4 subtypes (mixed, cholesterogenic, glycolytic, and quiescent) in an integrated cohort including TCGA, GSE13507, and IMvigor210. Clinical, genomic, transcriptomic, and tumor microenvironment characteristics were compared between the 4 subtypes.ResultsThe 4 metabolic subtypes exhibited distinct clinical, molecular, and genomic patterns. Compared to quiescent subtype, mixed subtype was more likely to be basal tumors and was significantly associated with poorer prognosis even after controlling for age, gender, histological grade, clinical stage, and molecular phenotypes. Additionally, mixed tumors harbored a higher frequency of RB1 and LRP1B copy number deletion compared to quiescent tumors (25.7% vs. 12.7 and 27.9% vs. 10.2%, respectively, both adjusted P value< 0.05). Furthermore, aberrant PIK3CA expression level was significantly correlated with those of glycolytic and cholesterogenic genes. The quiescent subtype was associated with lower stemness indices and lower signature scores for gene sets involved in genomic instability, including DNA replication, DNA damage repair, mismatch repair, and homologous recombination genes. Moreover, quiescent tumors exhibited lower expression levels of pyruvate dehydrogenase kinases 1-3 (PDK1-3) than the other subtypes. In addition, distinct immune cell infiltration patterns were observed across the 4 metabolic subtypes, with greater infiltration of M0/M2 macrophages observed in glycolytic and mixed subtypes. However, no significant difference in immunotherapy response was observed across the 4 metabolic subtypes.ConclusionThis study proposed a new metabolic subtyping method for BCa based on genes involved in glycolysis and cholesterol synthesis pathways. Our findings may provide novel insight for the development of personalized subtype-specific treatment strategies targeting metabolic vulnerabilities.

A prospective multicentric study of risk-reducing salpingo-oophorectomy in BRCA mutation patients.

Background and aim of the work:BRCA1/2 are tumour-suppressor genes involved in DNA homologous recombination and ovarian cancer development. The study evaluated the risk of tumor cancer in women presenting the BRCA mutations.Methods:Risk-reducing surgery (RRS) was performed in 100 patients carrying BRCA1 (aged between 30-73 years, median age was 51 years) and BRCA 2 mutation (aged between 36-70 years, median age was 53 years). Fifty-eight patients had previous history of breast cancer.Results:Between the 100 patients, 82 women underwent risk-reducing salpingo-oophorectomy (RRSO) through a laparoscopic minimally invasive approach, 7 (7%) underwent laparoscopic RRSO and contextual hysterectomy, 1 woman (1%) underwent RRSO through a laparotomic approach and 10 women (10%) laparotomic RRSO and hysterectomy. During 5 (5%) laparoscopic RRSO, prophylactic bilateral mastectomy was also performed. Early and late complication occurred in 3 patients (3%). Two patients (2%) were found to have occult Serous Tubal Intraepithelial Carcinoma (STIC) and three patients (3%) occult cancer.Conclusions:RRSO is safe and feasible in BRCA mutation carriers. The procedure is effective for genetic prevention of ovarian cancer. (www.actabiomedica.it)

LncRNA SNHG12 Inhibition Based on Microsystem Cell Imaging Technology Protects the Endothelium from LPS-Induced Inflammation by Inhibiting the Expression of miR-140-3p Target Gene fndc5.

Acute lung injury (ALI) is a serious disease with a high incidence rate, characterized by uncontrolled inflammation and apoptosis. At present, long-chain noncoding RNA (lncRNA) is a noncoding RNA with a length of more than 200 nucleotides. It plays an important role in ALI, cell cycle regulation, cell differentiation regulation, and many other life activities. Therefore, the current focus is to identify and evaluate the possible functions and potential molecular mechanisms of lncRNA small nuclear host gene 12 (SNHG12). Lipopolysaccharide (LPS)-induced mice model and in vitro cell model were established. Gene knockout is to use the principle of DNA homologous recombination to replace the target gene fragment with the designed homologous fragment, so as to achieve the purpose of gene knockout. The relationship between lncRNA SNHG12 expression and ALI was studied through knockdown and overexpression experiments. The qRT-PCR, ROS, immunohistochemistry, histopathology, TUNEL, and cell permeability tests were performed to further verify the possible targets and mechanisms of action. The expression of lncRNA SNHG12 in lung tissue was lower than that in normal tissue. The results showed that lncRNA SNHG12 could reduce lung cell injury and inflammatory cytokines induced by ALI. Bioinformatics analysis showed that lncRNA SNHG12 interacted with miR-140-3p. Subsequent experiments confirmed the link between lncRNA SNHG12, miR-140-3p, and fndc5. Furthermore, this study indicates that lncRNA SNHG12 has a key function in ALI. The results of this study demonstrated the role of lncRNA SNHG12 in the pathological process of ALI and provided a reference for developing novel anti-ALI treatments so that patients can get timely treatment, avoid causing multiple organ failure, and will not endanger their life safety.

Proguanil synergistically sensitizes ovarian cancer cells to olaparib by increasing DNA damage and inducing apoptosis.

Ovarian cancer is the second leading cause of cancer-related deaths in women, with low five-year survival rates. Therefore, it is essential to seek new treatment options. Olaparib, a PARP inhibitor, has benefited many ovarian cancer patients, but olaparib is much less effective as a single agent in 50% of patients with high grade severe tumors. Proguanil, which was originally developed as an anti-malarial drug, has gained attention due to its anti-tumor effects. Here, we evaluated the anti-tumor effect of the combination of olaparib and proguanil on ovarian cancer cells, aimed to develop a potential medical option for treating ovarian cancer patients. We examined the effect on proliferation by MTT and colony formation assays, while cell migration was measured by the transwell assay. The effect on apoptosis was measured by flow cytometry and AO/EB staining assays. Western blotting was used to detect protein expression levels in cells treated with olaparib and/or proguanil. In addition, the synergistic effect of these two drugs is calculated by CompuSyn software. The combination of olaparib and proguanil significantly increased growth suppression and apoptosis in ovarian cancer cells, compared to either single agent alone. Furthermore, results showed that the combination of olaparib and proguanil synergistically increased olaparib-induced apoptosis and DNA damage and reduced the efficiency of DNA homologous recombination repair. Our findings indicate that combination of olaparib with proguanil will be a novel potential administration route for treating ovarian cancer patients.

Opportunities for Utilization of DNA Repair Inhibitors in Homologous Recombination Repair-Deficient and Proficient Pancreatic Adenocarcinoma.

Pancreatic cancer is rapidly progressive and notoriously difficult to treat with cytotoxic chemotherapy and targeted agents. Recent demonstration of the efficacy of maintenance PARP inhibition in germline BRCA mutated pancreatic cancer has raised hopes that increased understanding of the DNA damage response pathway will lead to new therapies in both homologous recombination (HR) repair-deficient and proficient pancreatic cancer. Here, we review the potential mechanisms of exploiting HR deficiency, replicative stress, and DNA damage-mediated immune activation through targeted inhibition of DNA repair regulatory proteins.

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene

BackgroundFamilial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes.MethodsWhole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively.ResultsIn FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7–19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples.ConclusionsThis is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families.

Abstract P053: VAL-083 (dianhydrogalactitol) synergizes with PARP inhibitors in BRCA-proficient and BRCA-deficient ovarian cancer models

Abstract Platinum (Pt)-based chemotherapy plays a key role in ovarian cancer treatment, but patients frequently develop Pt-resistance. Dysfunctional p53 is implicated in Pt-resistance, comprising a therapeutic challenge in high grade serous ovarian carcinoma (HGSOC), where p53 is universally mutated. Attempts to overcome Pt-resistance in HGSOC include agents blocking the DNA repair pathways, most notably PARP inhibitors (PARPi), leading to accumulation of DNA double strand breaks (DSBs) and cancer cell death. Sensitivity to PARPi is correlated with deficiencies in the homologous recombination (HR) DNA repair system, which accurately repairs DSBs. BRCA1 and BRCA2 are key proteins in HR, and mutated BRCA1/2 are well-established biomarkers for PARPi sensitivity. PARPi treatment of Pt-sensitive ovarian cancers have improved progression free survival; however, improvements in overall survival have not been achieved and a 5-year survival rate of 40% remains in ovarian cancer. Additionally, resistance to PARPi has emerged as a significant clinical challenge. VAL-083 is a first-in-class bifunctional DNA damaging agent with demonstrated clinical activity against a range of cancers, including ovarian. VAL-083 rapidly induces interstrand cross-links at guanine-N7 leading to DSBs, activation of the HR repair pathway and cancer cell death. VAL-083 is able to overcome cisplatin-resistance in a panel of ovarian cancer in vitro models. We have also shown that VAL-083 maintains activity independent of prominent DNA repair mechanisms implicated in resistance to numerous chemotherapeutics including cisplatin and PARPi such as MGMT, non-homologous end-joining and mismatch repair. Cancer cells thus rely heavily on a functional HR pathway for repair of VAL-083-induced DSBs. This rationalizes VAL-083 combination therapy with agents inducing DSBs or blocking their repair, including PARPi. Taken together, these data highlight VAL-083's potential for targeting Pt-resistant HGSOC in combination with PARPi. METHODS: The cytotoxicity of VAL-083 in combination with PARPi (olaparib, niraparib, rucaparib, veliparib or talazoparib) against HR-impaired (BRCA siRNA knockdown) and HR-proficient (control siRNA) ovarian cancer cells was studied. RESULTS: We report increased VAL-083 cytotoxicity against HR-impaired A2780 cancer cells. In addition, synergy between VAL-083 and olaparib, talazoparib and niraparib in both HR-proficient and deficient settings. VAL-083 combination with rucaparib produced synergistic cytotoxicity in the HR-deficient setting, while VAL-083 combination with veliparib was additive. These data demonstrate that VAL-083 can synergize with some PARPi in both HR-proficient and HR-deficient settings. CONCLUSION: Our results demonstrate a distinct DNA-damaging mechanism for VAL-083, resulting in the ability to overcome cisplatin-resistance in HR-impaired tumors. In addition, VAL-083 synergized with several PARPi, particularly olaparib, rucaparib and talazoparib, in both HR-proficient and deficient ovarian tumor cells. Citation Format: Anne Steino, Guangan He, Beibei Zhai, Jeffrey Bacha, Dennis M. Brown, Mads Daugaard, Zahid H. Siddik. VAL-083 (dianhydrogalactitol) synergizes with PARP inhibitors in BRCA-proficient and BRCA-deficient ovarian cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P053.

Abstract P081: In vitro activity and efficacy of novel dual PARP-HDAC inhibitors

Abstract Introduction: Inhibition of poly adenosine diphosphate-ribose polymerase (PARP) is an effective treatment strategy against tumors with homologous recombination (HR) DNA repair deficiencies. Combination treatments utilizing PARP inhibitors (PARPi) in concert with inhibitors of key DNA repair or PARPi-resistance pathways, may expand the utility of PARPi beyond cancers harboring germ-line HR deficiencies. Acetylation and deacetylation of histones is an important regulatory event in the DNA damage response. Combining histone deacetylation inhibitors (HDACi) and PARPi have sensitized PARPi-resistant cells to treatment; however, combination regimens often require sequential administration to manage overlapping toxicities and accommodate diverse pharmacokinetics, constituting a significant limitation of these strategies in a clinical setting. Here, we test the activity of kt-3000 series, a novel bi-functional class of small molecules with dual PARP and HDAC inhibitor activity in HR-proficient cancer cells where PARPi have historically lacked single-agent activity. Methods: PARP enzyme activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit. HDAC activity was measured using nuclear extracts of treated cells and the HDAC Fluorometric Activity Assay Kit from Cayman Chemical, as per the manufacturer’s protocol. PARP activity was measured by fluorescence in C41 cells following compound treatment for 2 hours and PARP-activation with 1M H2O2 by staining cells with an anti-PARP Ab followed by a FITC-coupled secondary antibodies. Cell survival EC50 values were obtained by treating cells with a range of inhibitor concentrations, then quantifying cell confluency after 72-hours of treatment based on images taken with an Incucyte S3 system. Results: kt-3000 series compounds are potent inhibitors of PARP and HDAC with IC50 values for PARP enzyme activity in the low nM range, comparable to those of FDA-approved PARP inhibitors (olaparib, rucaparib, niraparib, and talazoparib) and IC50 values for the inhibition of HDAC activity comparable to FDA-approved HDAC inhibitors (panabinostat, belinostat, and vorinostat). Fluorescent assays of PARP activity in C41 cells also resulted in IC50 values in the nM range and cell survival EC50 values of the dual inhibitors are comparable to FDA-approved PARP inhibitors alone and in similar range with FDA-approved HDAC inhibitors alone. Conclusion: Our novel dual PARP-HDAC inhibitors show potent inhibition of PARP activity in vitro comparable to FDA-approved PARP inhibitors and also show potent inhibition of HDAC activity. The potency and activity of kt-3000 series compounds exhibit potential superiority to FDA-approved PARP inhibitors and HDAC inhibitors, in formulation as a single molecule. Development of these multi-target inhibitors will target unmet medical needs in the treatment of HR-proficient cancer types with dysregulation of histone deacetylation. Citation Format: Sarah Truong, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Mads Daugaard. In vitro activity and efficacy of novel dual PARP-HDAC inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P081.

Pathogenic variants of meiotic double strand break (DSB) formation genes PRDM9 and ANKRD31 in premature ovarian insufficiency

PurposeThe etiology of premature ovarian insufficiency (POI) is heterogeneous, and genetic factors account for 20–25% of the patients. The primordial follicle pool is determined by the meiosis process, which is initiated by programmed DNA double strand breaks (DSB) and homologous recombination. The objective of the study is to explore the role of DSB formation genes in POI pathogenesis.MethodsVariants in DSB formation genes were analyzed from a database of exome sequencing in 1,030 patients with POI. The pathogenic effects of the potentially causative variants were verified by further functional studies.ResultsThree pathogenic heterozygous variants in PRDM9 and two in ANKRD31 were identified in seven patients. Functional studies showed the variants in PRDM9 impaired its methyltransferase activity, and the ANKRD31 variations disturbed its interaction with another DSB formation factor REC114 by haploinsufficiency effect, indicating the pathogenic effects of the two genes on ovarian function were dosage dependent.ConclusionOur study identified pathogenic variants of PRDM9 and ANKRD31 in POI patients, shedding new light on the contribution of meiotic DSB formation genes in ovarian development, further expanding the genetic architecture of POI.

Radiosensitizing effect of c-Met kinase inhibitor BPI-9016M in esophageal squamous cell carcinoma cells in vitro and in vivo.

Backgroundc-Met is the receptor of hepatocyte growth factor (HGF) which plays a key role in inhibiting apoptosis. BPI-9016M is a small-molecule c-Met inhibitor that can promote apoptosis and enhance the cytotoxicity of various DNA-damaging agents. Here, we evaluated the radiosensitizing potential of BPI-9016M in Eca109 human esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo.MethodsCell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Clonogenic survival assay and a murine tumor xenograft in male nude mice were used to evaluate the radiosensitizing effect of BPI-9016M. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry experiment. Apoptosis-related proteins were detected by western blot. By evaluating the activation of the ATR-Chk1/ATM-Chk2 pathway to detect radiation-induced DNA double-strand break and homologous recombination repair. BPI-9016M induced a radiosensitizing effect in Eca109 cells and reduced the survival rate of clone formation in vitro.ResultsThe combination of BPI-9016M with irradiation (IR) significantly delayed the growth of ESCC tumor xenografts than treatment alone (P<0.05). The radiosensitizing effects of BPI-9016M were due to increased apoptosis, such as the up-regulation of cleaved-caspase 3 and 9, down-regulation of mutant P53 and Bcl-2, the decreased of phosphorylation of ATR and ATM, and the inhibition of γ-H2AX accumulation in vitro and in vivo.ConclusionsThese findings indicated that BPI-9016M exerts a radiosensitizing effect and enhances apoptosis by inhibiting homologous recombination DNA repair in irradiated ESCC cells.

QRT-PCR-based DNA homologous recombination-associated 4-gene score predicts pathologic complete response to platinum-based neoadjuvant chemotherapy in triple-negative breast cancer.

Cumulative evidence suggests that the addition of platinum agents as neoadjuvant chemotherapy (NACT) could improve the pathologic complete response (pCR) rate in triple-negative breast cancer (TNBC). We aimed to develop a DNA homologous recombination (HR)-associated gene expression score to predict tumor sensitivity to platinum-based NACT in TNBC. A retrospective cohort of 127 patients who were diagnosed with TNBC and received platinum-based NACT in Fudan University Shanghai Cancer Center from 2012 to 2017 was included in this study. Using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the expression levels of eight HR-associated genes were analyzed from formalin-fixed paraffin-embedded core-needle biopsy samples obtained before NACT. A random forest model was built to estimate the weight of each gene expression level and clinicopathological factors. The training set was used to modulate parameters and select the best model. The performance of the final model was evaluated in the validation set. A 4-gene (BRCA1, XRCC5, PARP1, and RAD51) scoring system was developed. TNBC patients with a higher score had a nearly fourfold likelihood of achieving pCR to platinum-based NACT compared with patients with a lower score [odds ratio (OR) = 3.878; P < 0.001]. At the cutoff value of -2.644, the 4-gene scoring system showed high sensitivity in predicting pCR in the breast (93.0%) and pCR in the breast/axilla (91.8%), while at the cutoff value of -1.969, the 4-gene score showed high specificity for pCR in the breast (85.7%) and pCR in the breast/axilla (80.8%). The qRT-PCR-based 4-gene score has the potential to predict pCR to platinum-based NACT in TNBC.

Germinal &lt;i&gt;BRCA&lt;/i&gt;-mutation significance in the tumor microenvironment formation Efficacy of PARP inhibition in late-line therapy of metastatic castration-resistant prostate cancer

Metastatic castration-resistant prostate cancer is a difficult problem for a clinical oncologist. In addition, mutations in genes of homologous DNA recombination, including BRCA1/2, suggest an aggressive behavior and therapy resistance. Treatment options for such patients were significantly limited until new drugs - PARP inhibitors have been registered. Nevertheless, there is evidence that BRCA1/2 gene mutations are associated with increased mutational load, neoepitopes formation, increased number of tumor-infiltrating lymphocytes and a response to the immune response checkpoints blockade. Studies have shown that BRCA2-mutated prostate cancer demonstrates high level of immune cells infiltration compared to tumors without mutation, in particular with respect to CD4+, CD8+ and FOXP3+ T-lymphocytes. It should be noted that studies have shown a tendency of CD8+ T-lymphocytes/FOXP3+ T-cells ratio decreasing in BRCA2-mutated tumors. Thus, the mutational status of BRCA2 presumably forms the immune phenotype of prostate cancer with an increase of intratumoral immune cells, but with immunosuppressive properties. At the same time, the use of immune checkpoint blockers in advanced prostate cancer has been unsuccessful in terms of overall survival. Despite the fact that immune checkpoint blocker's efficacy is often associated with a high intracellular CD4+ and CD8+ T lymphocytes, their presence is clearly insufficient for response. Studies showed that PARP inhibitors effect tumor microenvironment significantly. Anti-PD-1/PD-L1 combination with PARP inhibitors is being actively studied due to their properties of modulating the tumor microenvironment. Thus, future immunooncological strategies for primary prostate cancer therapy may include not only an increase in mutational load, but also an impact on the immunosuppressive microenvironment. The article presents clinical cases of 3 brothers, carriers of the germinal BRCA2 c.9371A&gt;T mutation, suffering from prostate cancer with a burdened family history. The disease development under standard therapies was studied and markers of the tumor microenvironment were immunohistochemically evaluated. PARP inhibitor Olaparib efficacy in prostate cancer of older brother in late-line therapy for metastatic castration-resistant disease was analyzed.

Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.

PPIP5K2 promotes colorectal carcinoma pathogenesis through facilitating DNA homologous recombination repair.

Colorectal carcinoma (CRC) is the second most deadly cancer worldwide. Therapies that take advantage of DNA repair defects have been explored in various tumors but not yet systematically in CRC. Here, we found that Diphosphoinositol Pentakisphosphate Kinase 2 (PPIP5K2), an inositol pyrophosphate kinase, was highly expressed in CRC and associated with a poor prognosis of CRC patients. In vitro and in vivo functional studies demonstrated that PPIP5K2 could promote the proliferation and migration ability of CRC cells independent of its inositol pyrophosphate kinase activity. Mechanically, S1006 dephosphorylation of PPIP5K2 could accelerate its dissociation with 14-3-3 in the cytoplasm, resulting in more nuclear distribution. Moreover, DNA damage treatments such as doxorubicin (DOX) or irradiation (IR) could induce nuclear translocation of PPIP5K2, which subsequently promoted homologous recombination (HR) repair by binding and recruiting RPA70 to the DNA damage site as a novel scaffold protein. Importantly, we verified that S1006 dephosphorylation of PPIP5K2 could significantly enhance the DNA repair ability of CRC cells through a series of DNA repair phenotype assays. In conclusion, PPIP5K2 is critical for enhancing the survival of CRC cells via facilitating DNA HR repair. Our findings revealed an unrecognized biological function and mechanism model of PPIP5K2 dependent on S1006 phosphorylation and provided a potential therapeutic target for CRC patients.

The Effect of Atypical Nucleic Acids Structures in DNA Double Strand Break Repair: A Tale of R-loops and G-Quadruplexes.

The fine tuning of the DNA double strand break repair pathway choice relies on different regulatory layers that respond to environmental and local cues. Among them, the presence of non-canonical nucleic acids structures seems to create challenges for the repair of nearby DNA double strand breaks. In this review, we focus on the recently published effects of G-quadruplexes and R-loops on DNA end resection and homologous recombination. Finally, we hypothesized a connection between those two atypical DNA structures in inhibiting the DNA end resection step of HR.