Homologous Recombination Pathway Research Articles

Inhibition of PKM2 Enhances Sensitivity of Olaparib to Ovarian Cancer Cells and Induces DNA Damage.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have showed clinical benefit as maintenance therapy in advanced ovarian cancer by impairing the homologous recombination (HR) pathway. Pyruvate kinase M2 (PKM2), the significant cancer metabolic biomarker, integrates with DNA damage to directly promote HR. We aimed to investigate the role and molecular mechanism of PKM2 downregulation on sensitization of ovarian cancer cells to PARPi. Inhibitory effects in vitro were assessed by cell viability, clone formation, transwell assay, and flow cytometry. Downregulation of PKM2 by siRNA or small molecular inhibitor shikonin (Sk) enhanced anti-tumour activity of olaparib (Ola) in ovarian cancer cells. Silencing PKM2 or Sk synergized with Ola and reduced cell growth, colony formation and migration, and induced apoptosis. Western blot and immunofluorescence demonstrated that inhibition of PKM2 amplified Ola-induced γH2AX and phospho-ATM (p-ATM) activation and interfered with BRCA1 accumulation in the nucleus. A xenograft animal model demonstrated in vivo antitumor combination effect of Sk and Ola. Furthermore, Western blot and immunofluorenscent analyses of tissue samples revealed that treatment of Sk increased DNA damage, reduced expression of BRCA1 and PKM2. Therefore, this study identified that PKM2 downregulation is a novel therapeutic strategy to enhance Ola effectiveness in treating ovarian cancer.

Homologous Recombination Deficiencies and Hereditary Tumors.

Homologous recombination (HR) is a vital process for repairing DNA double-strand breaks. Germline variants in the HR pathway, comprising at least 10 genes, such as BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK2, NBS1(NBN), PALB2, RAD51C, and RAD51D, lead to inherited susceptibility to specific types of cancers, including those of the breast, ovaries, prostate, and pancreas. The penetrance of germline pathogenic variants of each gene varies, whereas all their associated protein products are indispensable for maintaining a high-fidelity DNA repair system by HR. The present review summarizes the basic molecular mechanisms and components that collectively play a role in maintaining genomic integrity against DNA double-strand damage and their clinical implications on each type of hereditary tumor.

Panobinostat enhances olaparib efficacy by modifying expression of homologous recombination repair and immune transcripts in ovarian cancer.

Histone deacetylase inhibitors (HDACi) sensitize homologous recombination (HR)-proficient human ovarian cancer cells to PARP inhibitors (PARPi). To investigate mechanisms of anti-tumor effects of combined HDACi/PARPi treatment we performed transcriptome analysis in HR- proficient human ovarian cancer cells and tested drug effects in established immunocompetent mouse ovarian cancer models. Human SKOV-3 cells were treated with vehicle (Con), olaparib (Ola), panobinostat (Pano) or Pano+Ola and RNA-seq analysis performed. DESeq2 identified differentially expressed HR repair and immune transcripts. Luciferised syngeneic mouse ovarian cancer cells (ID8-luc) were treated with the HDACi panobinostat alone or combined with olaparib and effects on cell viability, apoptosis, DNA damage and HR efficiency determined. C57BL/6 mice with intraperitoneally injected ID8-luc cells were treated with panobinostat and/or olaparib followed by assessment of tumor burden, markers of cell proliferation, apoptosis and DNA damage, tumor-infiltrating T cells and macrophages, and other immune cell populations in ascites fluid. There was a significant reduction in expression of 20/37 HR pathway genes by Pano+Ola, with immune and inflammatory-related pathways also significantly enriched by the combination. In ID8 cells, Pano+Ola decreased cell viability, HR repair, and enhanced DNA damage. Pano+Ola also co-operatively reduced tumor burden and proliferation, increased tumor apoptosis and DNA damage, enhanced infiltration of CD8+ T cells into tumors, and decreased expression of M2-like macrophage markers. In conclusion, panobinostat in combination with olaparib targets ovarian tumors through both direct cytotoxic and indirect immune-modulating effects.

SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites.

DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.

Mise à jour 2021 des recommandations pour la pratique clinique de Nice/Saint-Paul-de-Vence dans le cancer de l’ovaire épithélial de haut grade: Updated 2021 recommendations for the clinical practice of Nice/Saint-Paul-de-Vence in epithelial high grade ovarian cancer

Since the previous 2013 and 2016 recommendations for clinical practice (RPC) Nice/Saint-Paul-de-Vence for gynecological cancers, the management of ovarian cancer has become more complex with the evolution of the quality criteria recommended for surgery and the integration of molecular biology for the decision of medical treatments, especially for high grade epithelial ovarian cancers. Surgical indications have become more precise both in the first line and in the context of relapse. Treatments with PARP inhibitors is a major advance in medical management with significant efficacy in maintenance after response to platinum-based chemotherapy. The benefit already known in the case of late relapse has also been demonstrated in first-line treatment with progression-free survival never observed in this pathology with patients with very long responses, especially in the case of BRCA gene abnormalities (somatic or constitutional). In 2021, medical and surgical strategies in front line including PARP inhibitors associated or not with bevacizumab as a maintenance complement after platinum chemotherapy are guided by both response to platinum agents and molecular profiling including BRCA (somatic or constitutional) genetic status and homologous recombination pathway (HRD) abnormalities, that should be early tested. On behalf of the GINECO national oncologist group, we have updated the guidelines for high grade ovarian epithelial cancer (excepted rare tumors) in order to allow rapid dissemination of the latest advances to the medical community and improve daily practice.

Abstract LBA041: Identification and characterization of potent RAD51 inhibitors targeting RAD51-BRCA2 interaction

Abstract The DNA damage response (DDR) is a complex cellular network that acts as a cell defense mechanism and exerts signal for repair of DNA lesions. It repairs different types of DNA damage such as single-strand (ssDNA) breaks, DNA inter-strand crosslinks and double strand breaks (DSB) caused by endogenous and exogenous factors. These damages are mainly repaired by homologous recombination (HR) repair, and non-homologous end joining (NHEJ). DNA double-strand breaks (DSBs) are the most severe DNA damage and predominantly repaired by HR. As the DDR pathway is critical for tumorigenesis, a high degree of interest has been generated in the recent past to target this network. RAD51 is one of the pivotal enzymes for DNA double-strand break repair by the HR pathway. While PARP inhibitors are extensively used for BRCA2 mutated cancers, their responses in BRCA2 WT is limited. Targeting RAD51 will open a new avenue to treat BRCA2 WT cancer patients. BRCA2 interaction with RAD51 is a prerequisite for RAD51 nuclear translocation to exert its function by binding to DNA. The disruption of this interaction leads to inhibition of DNA repair and in turn inhibits proliferation and tumor growth. As this leads to BRCAness in an otherwise BRCA2 WT background, it is expected to have synergy with PARP1 inhibitors. We report the identification of novel and potent RAD51 inhibitors which disrupt the RAD51:BRCA2 interaction. Our hit compounds show anticancer activity in relevant cancer cell lines across various indications. Further, our compounds inhibit RAD51 nuclear translocation upon induction of exogenous DNA damage. Multiple series have been identified, with a good SAR trend and correlating well with cell based and PD activity. Further profiling of these compounds is ongoing. Citation Format: Sanjita Sasmal, Sukanya Patra, Venkatesham Boorgu, Mahesh Yanamandra, Shankar Chithaluri, Hema Sankar Pathange, Deepika Hiremath, Sandeep M Girme, Amit Kumar Pawar. Identification and characterization of potent RAD51 inhibitors targeting RAD51-BRCA2 interaction [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 LBA041.

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.

Dynamically Accumulating Homologous Recombination Deficiency Score Served as an Important Prognosis Factor in High-Grade Serous Ovarian Cancer.

Background: The homologous recombination (HR) pathway defects in cancers induced abrogation of cell cycle checkpoints, resulting in the accumulation of DNA damage, mitotic catastrophe, and cell death. Cancers with BRCA1/2 loss and other accumulation of similar genomic scars resulting in HRD displayed increased sensitivity to chemotherapy. Our study aimed to explore HRD score genetic mechanisms and subsequent clinical outcomes in human cancers, especially ovarian cancer. Methods: We analyzed TCGA data of HRD score in 33 cancer types and evaluated HRD score distribution and difference among tumor stages and between primary and recurrent tumor tissues. A weighted gene co-expression network analysis (WGCNA) was performed to identify highly correlated genes representing essential modules contributing to the HRD score and distinguish the hub genes and significant pathways. We verified HRD status predicting roles in patients’ overall survival (OS) with univariate and multivariate Cox regression analyses and built the predicting model for patient survival. Results: We found that the HRD score increased with the rise in tumor stage, except for stage IV. The HRD score tended to grow up higher in recurrent tumor tissue than in their primary counterparts (p = 0.083). We constructed 15 co-expression modules with WGCNA, identified co-expressed genes and pathways impacting the HRD score, and concluded that the HRD score was tightly associated with tumor cells replication and proliferation. A combined HRD score ≥42 was associated with shorter OS in 33 cancer types (HR = 1.010, 95% CI: 1.008–1.011, p < 0.001). However, in ovarian cancer, which ranked the highest HRD score among other cancers, HRD ≥42 cohort was significantly associated with longer OS (HR = 0.99, 95% CI: 0.98–0.99, p < 0.0001). We also built a predicting model for 3 and 5 years survival in HGSC patients. Conclusion: A quantitative HRD score representing the accumulated genomic scars was dynamically increasing in proliferating tumor cells since the HRD score was tightly correlated to tumor cell division and replication. We highlighted HRD score biomarker role in prognosis prediction of ovarian cancer.

Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

Place des inhibiteurs de PARP dans le traitement des cancers de l’endomètre et du col de l’utérus

New molecular therapeutic approaches have emerged in recent years for advanced gynaecological cancers, including targeted therapies such as poly-ADP-ribose polymerase inhibitors (PARPi). These have demonstrated efficacy in high-grade serous ovarian cancers in patients carrying a mutation in the BRCA gene, which predisposes them to breast and ovarian cancers. Clinical and pre-clinical data suggest that the activity of PARPi inhibitors may not be limited to BRCA mutated tumours and may involve the homologous recombination pathway. These data raise the question of the potential efficacy of PARPi in advanced endometrial and cervical cancers where treatment options are currently limited. At present, there are few data available on the activity of PARPi in endometrial and cervical cancers, but some results seem promising. In this review, we present a synthesis of the available studies concerning PARPi in endometrial and cervical cancer.

Loss of PTEN in Pediatric AML Confers Sensitivity to PARP Inhibition

Pediatric Acute Myeloid Leukemia (AML) is a rare, but deadly cancer. Outcomes over the last 20 years have remained stagnant with an overall 5-year survival rate < 70% and relapse rates around 50%. Further, few new therapies have been successfully introduced to improve these outcomes. Here we report that exploiting deficiencies in DNA damage repair (DDR) is a potential therapeutic strategy for AML. Poly-ADP Ribose Polymerase (PARP) inhibitors were initially developed to target deficient homologous recombination (HR) in BRCA1/2 mutated cancers by blocking single stranded base repair following DNA damage, leading to an accumulation of double stranded DNA breaks, thereby inducing apoptosis.To evaluate the activity of PARP inhibition in pediatric AML, talazoparib was tested as a single agent and in combination with standard chemotherapeutic agents in human AML cell lines representing low (Kasumi-1 and ME-1), intermediate (AML193), and high-risk (CTS, CMS, MOLM-13, and CHRF288-11) disease based on their genomic mutations. Talazoparib showed the highest efficacy as a single agent in all four cell lines with genomic lesions found in high-risk AML subtypes.After combination drug screens, topotecan (synergistic) and gemcitabine (additive) were chosen to move forward to in vivo testing. Our investigational combination was tested in vivo in four murine models representing pediatric AML subtypes harboring AML1-ETO9a (low risk), MLL-AF6 (high risk), CBAF2T3-GLIS2/JAK2 V617F (high risk) and NUP98-KDM5A (high risk) oncogenes. Mice received a backbone of either current standard of care chemotherapy (SOC; anthracycline plus cytarabine) or topotecan plus gemcitabine. NUP98-KDM5A and MLL-AF6 positive mice receiving single agent talazoparib were found to have prolonged survival compared to vehicle alone (p=0.019 and p<0.0001, respectively) which was further enhanced by the addition of chemotherapy irrespective of backbone (p <0.0001). Conversely, mice with AML1-ETOa positive leukemia had no response to single agent PARP inhibitor. While a few mice benefitted from the addition of talazoparib to SOC, this result was not statistically significant (p= 0.42). Early response by bioluminescent imaging confirmed that mice with MLL-AF6 and NUP98-KDM5A driven leukemias who received talazoparib in combination with chemotherapy had the lowest leukemia burdens while the AML1-ETOa cohort did not benefit from the addition of this targeted agent. Interestingly, mice harboring CBAF2T3-GLIS2/JAK2 V617F were not responsive to PARP inhibitors, which was inconsistent with the CMS cell line that has same oncogenic fusion gene but lacks the JAK2 V617F mutation.Synergy experiments with ATM inhibitor AZD0156 demonstrated tremendous synergy with talazoparib in sensitive cell lines with almost no synergy in those that were resistant, suggesting that sensitive cell lines are unable to efficiently activate the HR pathway to repair double stranded breaks induced by PARP inhibition whereas resistant cells can overcome inhibition. To determine the HR response to DNA damage in our cell lines, we exposed them to 1uM topotecan for 2 hours and then measured γH2AX response at 0, 4 and 24 hours. γH2AX is a sensor of DNA damage and therefore increases with DNA damage and decreases with repair. PARP inhibitor sensitive cell lines had persistence of gamma H2AX at 24hrs while resistant cell lines had at least partial resolution of damage, confirming that PARP inhibitor sensitive cell lines have aberrant DNA damage response through HR.RNA sequencing of our cell lines revealed a correlation between Phosphatase and tensin homolog (PTEN) transcript levels and PARP sensitivity. Western blotting confirmed that PTEN was downregulated or absent in both cell lines and murine leukemias that were sensitive to PARP inhibitors. In contrast to the CMS cell line that carries the CBFA2T3-GLIS2 fusion, murine leukemias with CBAF2T3-GLIS2/JAK2 V617F had high levels of PTEN, supporting the hypothesis that sensitivity to PARP inhibitors is due to loss of PTEN.In conclusion, we report that a subset of pediatric AML with high- risk features are sensitive to PARP inhibition due to deficient DDR through HR. Downregulation of PTEN is a candidate biomarker of response to PARP inhibitors in these patients. This data illuminates a promising therapeutic vulnerability in a patient population where new targeted treatments are vital to improve outcomes. DisclosuresNo relevant conflicts of interest to declare.

TRIM33 Loss in Multiple Myeloma Impairs the DNA Damage Response Resulting in Sensitivity to PARP and ATR Inhibitors

IntroductionChromosomal instability is a hallmark of Multiple Myeloma (MM), with most patients displaying cytogenetic abnormalities which can arise due to DNA damage response (DDR) defects. TRIM33 is an E3 ligase and transcriptional co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 plays a role in the DDR and can regulate chromosomal stability, but its precise function remains unknown. In this study we investigated the impact of TRIM33 loss in MM on genomic stability and DDR pathways and whether this could be exploited therapeutically.MethodsThe CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with overall survival (OS) and structural variants. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells. The effect on DDR signalling was determined by western blotting and immunofluorescence. The Selleckchem DNA Damage/Repair Compound Library was screened on the JJN3 model in a high-throughput manner using the CellTox™ Green cytotoxicity assay. Validation of selected compounds was performed using CellTiter® Glo viability assay or clonogenic assays. Combination indices (CI) were calculated using CompuSyn software.ResultsData on CN, OS and structural variants were available for 730 newly diagnosed MM patients and of these, 69 (9.5%) were identified to have a CN loss of TRIM33. These patients have poorer OS compared to those without TRIM33 loss (52.3 months vs 72.6 months; p<0.0001). Moreover, they exhibit a significantly higher median number of structural variants (deletions, duplications, inversions, and translocations; 38 vs 26; p<0.0001), indicative of increased chromosomal instability.Our data in MM cell lines has shown that TRIM33 is rapidly recruited to chromatin within 5 minutes of induced DNA damage. TRIM33 knockdown led to an increase in 53BP1 foci formation and endogenous γH2AX (P<0.001) indicating unrepaired DNA double-strand breaks (DSBs) typical of a DDR defect. In response to these DSBs both ATM and ATR kinases were activated as demonstrated by increased pKAP1 Ser824 and pCHK1 Ser345 respectively (p<0.001). Additionally, we observed a reduction in RAD51 (p<0.05) indicative of a potential defect in the DSB repair pathway homologous recombination (HR).To identify therapeutic vulnerabilities relating to TRIM33 loss, we performed a high-throughput screen to assess sensitivity to 160 unique DNA damaging compounds. TRIM33 knockdown cells exhibited increased sensitivity to 27 compounds across a range of drug classes. Additional studies confirmed that compared to control cells, TRIM33 knockdown sensitized cells to the PARP inhibitor Olaparib and ATR inhibitors BAY-1895344 and VE-821. Further investigation with VE-821 demonstrated that whilst treatment induced PARP cleavage and DSBs in both control and knockdown cells within 48 hours, knockdown cells exhibited significantly more pCHK1 Ser345 inhibition (p<0.01). Furthermore, combining VE-821 with bortezomib yielded synergistic effects in TRIM33 knockdown cells across a range of doses (CI range 0.57-0.9) while no synergy was observed in control cells (CI>1 for all combinations).ConclusionWe have identified a subset of MM patients with TRIM33 loss who display high-risk disease characterized by chromosomal abnormalities and defective DDR. Alongside this we have identified PARP and ATR inhibitors as therapeutic vulnerabilities in cell line models of TRIM33 loss. Moreover, we demonstrate that ATR inhibition increases the efficacy of bortezomib in TRIM33 knockdown cells. Further investigation into these compounds could lead to novel therapies for patients with TRIM33 loss. DisclosuresNo relevant conflicts of interest to declare.

Bloom Helicase Along with Recombinase Rad51 Repairs the Mitochondrial Genome of the Malaria Parasite.

ABSTRACTThe homologous recombination (HR) pathway has been implicated as the predominant mechanism for the repair of chromosomal DNA double-strand breaks (DSBs) of the malarial parasite. Although the extrachromosomal mitochondrial genome of this parasite experiences a greater number of DSBs due to its close proximity to the electron transport chain, nothing is known about the proteins involved in the repair of the mitochondrial genome. We investigated the involvement of nucleus-encoded HR proteins in the repair of the mitochondrial genome, as this genome does not code for any DNA repair proteins. Here, we provide evidence that the nucleus-encoded “recombinosome” of the parasite is also involved in mitochondrial genome repair. First, two crucial HR proteins, namely, Plasmodium falciparum Rad51 (PfRad51) and P. falciparum Bloom helicase (PfBlm) are located in the mitochondria. They are recruited to the mitochondrial genome at the schizont stage, a stage that is prone to DSBs due to exposure to various endogenous and physiologic DNA-damaging agents. Second, the recruitment of these two proteins to the damaged mitochondrial genome coincides with the DNA repair kinetics. Moreover, both the proteins exit the mitochondrial DNA (mtDNA) once the genome is repaired. Most importantly, the specific chemical inhibitors of PfRad51 and PfBlm block the repair of UV-induced DSBs of the mitochondrial genome. Additionally, overexpression of these two proteins resulted in a kinetically faster repair. Given the essentiality of the mitochondrial genome, blocking its repair by inhibiting the HR pathway could offer a novel strategy for curbing malaria.IMPORTANCE The impact of malaria on global public health and the world economy continues to surge despite decades of vaccine research and drug development efforts. An alarming rise in resistance toward all the commercially available antimalarial drugs and the lack of an effective malaria vaccine brings us to the urge to identify novel intervention strategies for curbing malaria. Here, we uncover the molecular mechanism behind the repair of the most deleterious form of DNA lesions on the parasitic mitochondrial genome. Given that the single-copy mitochondrion is an indispensable organelle of the malaria parasite, we propose that targeting the mitochondrial DNA repair pathways should be exploited as a potential malaria control strategy. The establishment of the parasitic homologous recombination machinery as the predominant repair mechanism of the mitochondrial DNA double-strand breaks underscores the importance of this pathway as a novel druggable target.

Expression Levels of RAD51 Inversely Correlate with Survival of Glioblastoma Patients.

Simple SummaryIdentifying prognostic and predictive biomarkers for glioblastoma (GBM), a primary brain tumor, is essential in improving patient survival. We utilized gene expression profiling to investigate a uniform population of GBM patients who had been treated with surgery and adjuvant radiation therapy versus normal brain tissue, and identified high RAD51 expression as a poor prognostic marker that is amenable to therapeutic intervention. This observation was confirmed utilizing a publicly available gene expression dataset in a cohort of GBM patients.Treatment failures of glioblastoma (GBM) occur within high-dose radiation fields. We hypothesized that this is due to increased capacity for DNA damage repair in GBM. We identified 24 adult GBM patients treated with maximal safe resection followed by radiation with concurrent and adjuvant temozolomide. The mRNA from patients was quantified using NanoString Technologies’ nCounter platform and compared with 12 non-neoplastic temporal lobe tissue samples as a control. Differential expression analysis identified seven DNA repair genes significantly upregulated in GBM tissues relative to controls (>4-fold difference, adjusted p values < 0.001). Among these seven genes, Cox proportional hazards models identified RAD51 to be associated with an increased risk of death (HR = 3.49; p = 0.03). Kaplan–Meier (KM) analysis showed that patients with high RAD51 expression had significantly shorter OS compared to low levels (median OS of 10.6 mo. vs 20.1 mo.; log-rank p = 0.03). Our findings were validated in a larger external dataset of 162 patients using publicly available gene expression data quantified by the same NanoString technology (median OS of 13.8 mo. vs. 17.4 mo; log-rank p = 0.006). Within this uniformly treated GBM population, RAD51, in the homologous recombination pathway, was overexpressed (vs. normal brain) and inversely correlated with OS. High RAD51 expression may be a prognostic biomarker and a therapeutic target in GBM.

Downregulation of STOX1 is a novel prognostic biomarker for glioma patients.

Storkhead box 1 (STOX1) is a winged helix transcription factor structurally and functionally related to the forkhead family of transcription factors. Recent studies have highlighted its role in the central nervous system and revealed hints in the development of glioma. However, the expression profiles of STOX1, its association with clinicopathological characteristics, and potential functions in glioma remain unknown. In this study, we analyzed three publicly available datasets including CGGA, TCGA, and Rembrandt and revealed a grade-dependent reduction in STOX1 expression in glioma (P < 0.001). Chi-square test demonstrated that low STOX1 expression was significantly associated with older age at initial diagnosis (P < 0.001), less IDH1 mutation (P < 0.001), and advanced WHO grade (P < 0.001). Moreover, multivariate Cox regression analysis showed that STOX1 expression may serve as a novel independent prognostic biomarker in glioma patients. Bioinformatic functional analysis (GSEA) predicted that STOX1 was related to many key cancer pathways including P53 signaling pathway (P < 0.01), DNA replication (P < 0.05), homologous recombination (P < 0.05), and Wnt signaling pathway (P < 0.05). Taken together, these findings suggested that STOX1 may be used as a novel predictive molecular biomarker for glioma grading and overall patient survival. Further investigations on the functional roles and therapeutic value of STOX1 in glioma are warranted.

BET Proteins as Attractive Targets for Cancer Therapeutics.

Transcriptional dysregulation is a hallmark of cancer and can be an essential driver of cancer initiation and progression. Loss of transcriptional control can cause cancer cells to become dependent on certain regulators of gene expression. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that regulate the expression of multiple genes involved in carcinogenesis. BET inhibitors (BETis) disrupt BET protein binding to acetylated lysine residues of chromatin and suppress the transcription of various genes, including oncogenic transcription factors. Phase I and II clinical trials demonstrated BETis’ potential as anticancer drugs against solid tumours and haematological malignancies; however, their clinical success was limited as monotherapies. Emerging treatment-associated toxicities, drug resistance and a lack of predictive biomarkers limited BETis’ clinical progress. The preclinical evaluation demonstrated that BETis synergised with different classes of compounds, including DNA repair inhibitors, thus supporting further clinical development of BETis. The combination of BET and PARP inhibitors triggered synthetic lethality in cells with proficient homologous recombination. Mechanistic studies revealed that BETis targeted multiple essential homologous recombination pathway proteins, including RAD51, BRCA1 and CtIP. The exact mechanism of BETis’ anticancer action remains poorly understood; nevertheless, these agents provide a novel approach to epigenome and transcriptome anticancer therapy.

Testis transcriptome profiling identified lncRNAs involved in spermatogenic arrest of cattleyak

Cattleyaks are the crossbred offspring between cattle and yaks, exhibiting the prominent adaptability to the harsh environment as yaks and much higher growth performances than yaks around Qinghai-Tibet plateau. Unfortunately, cattleyak cannot be effectively used in yak breeding due to its male infertility resulted from spermatogenic arrest. In this study, we performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine the expression profiles of long noncoding RNA (lncRNA) from cattleyak and yak testis. A total of 604 differentially expressed (DE) lncRNAs (135 upregulated and 469 downregulated) were identified in cattleyak with respect to yak. Through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we identified several DE lncRNAs regulating the mitotic cell cycle processes by targeting the genes significantly associated with the mitotic cell cycle checkpoint and DNA damage checkpoint term and also significantly involved in p53 signaling pathway, mismatch repair and homologous recombination pathway (P < 0.05). The reverse transcription PCR (RT-PCR) and quantitative Real-Time PCR (qRT-PCR) analysis of the randomly selected fourteen DE lncRNAs and the seven target genes validated the RNA-seq data and their true expressions during spermatogenesis in vivo. Molecular cloning and sequencing indicated that the testis lncRNAs NONBTAT012170 and NONBTAT010258 presented higher similarity among different cattleyak and yak individuals. The downregulation of these target genes in cattleyak contributed to the abnormal DNA replication and spermatogenic arrest during the S phase of mitotic cell cycle. This study provided a novel insight into lncRNA expression profile changes associated with spermatogenic arrest of cattleyak.

Comprehensive Genetic Features of Serous Ovarian Tumor Patients Revealed PIK3CA Mutation and Chromosome Instability as Prognostic Biomarkers

Background: Ovarian cancer is the seventh most common cancer in women worldwide among which the most frequently occurred histological type is serous ovarian cancer (SOC). Since efficacious treatments for SOC have not advanced beyond platinum-based combination chemotherapy and more than 75% of high-grade SOC will relapse after first-line therapy, it is urgent to observe the genomic abnormalities and identify novel therapeutic targets and prognosis biomarkers. Methods: In order to comprehensively identify molecular features of serous ovarian cancer, we performed targeted sequencing with 425 cancer-related genes on four serous ovarian tumor (SOT) cohorts, classified as ovarian serous adenoma (OSA), ovarian serous borderline tumor (OSBT), low-grade serous cancer (LGSC) and high-grade serous cancer (HGSC). The association between genetic alterations and patients’ overall survival (OS) was analyzed. Results: Genomic profiling revealed distinct molecular features among these four cohorts. The frequency of genetic alterations in OSA was relatively low, and in OSBT cohort, the predominantly mutated genes, BRAF and KRAS, were identified at prevalence of 52.6% (10/19) and 36.8% (7/19) respectively with two patients harbored both these two mutations. In LGSC cohort, alterations of KRAS still occupied the highest percentage of patients which was up to 50.0% (5/10) while BRAF was not common (1/10, 10.0%). The most frequently mutated gene was TP53 in HGSC (46/47, 97.9%), whereas BRAF or KRAS mutation was rare. Meanwhile, a higher prevalence of gene copy gains in PTK2 (12/47, 25.5%), MYC (9/47, 19.1%), MDM4 (5/47, 10.6%) and ZNF217 (5/47, 10.6%) were identified only in HGSC group which indicated cancer progression promoted by chromosomal instability in this group. The median tumor mutational burden (TMB) and chromosome instability score (CIS) in cases with LGSC and HGSC higher than that in OSBT. Additionally, analysis of DNA damage repair (DDR) relevant genes showed most altered genes enriched in homologous recombination (HR) pathway in HGSC. Finally, we correlated genomic profiles with overall survival (OS) and found that PIK3CA wildtype or chromosome instability score (CIS) low patients had significantly longer OS in HGSC. Conclusion: In this study, we revealed the comprehensive genomic profiling among four SOT cohorts. Additionally, we correlated PIK3CA status and first associated chromosome instability with clinical outcomes of patients and found them to be useful clinical biomarkers in HGSC prognosis.

Downregulation of p38 MAPK Activation and Radiation-Sensitive 52 Expression Enhances 5-Fluorouracil and Erlotinib-Induced Cytotoxicity in Human Lung Squamous Cells

Introduction: 5-Fluorouracil (5-FU) is used to treat various cancers, including non-small-cell lung cancer (NSCLC). It inhibits nucleotide synthesis and induces single- and double-strand DNA breaks. In the homologous recombination pathway, radiation-sensitive 52 (Rad52) plays a crucial role in DNA repair by promoting the annealing of complementary single-stranded DNA and stimulating Rad51 recombinase activity. Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor with clinical activity against NSCLC cells. However, whether the combination of 5-FU and erlotinib has synergistic activity against NSCLC cells is unknown. Methods: After the 5-FU and/or erlotinib treatment, the expressions of Rad52 mRNA were determined by quantitative real-time polymerase chain reaction analysis. Protein levels of Rad52 and phospho-p38 MAPK were determined by Western blot analysis. We used specific Rad52 or p38 MAPK small interfering RNA and p38 MAPK inhibitor (SB2023580) to examine the role of p38 MAPK-Rad52 signal in regulating the chemosensitivity of 5-FU and/or erlotinib. Cell viability was assessed by MTS assay and trypan blue exclusion assay. Results: In 2 squamous cell carcinoma cell lines, namely, H520 and H1703, 5-FU reduced Rad52 expression in a p38 MAPK inactivation-dependent manner. Enhancement of p38 MAPK activity by transfection with MKK6E (a constitutively active form of MKK6) vector increased the Rad52 protein level and cell survival by 5-FU. However, in human lung bronchioloalveolar cell adenocarcinoma A549 cells, 5-FU reduced Rad52 expression and induced cytotoxicity independent of p38 MAPK. Moreover, 5-FU synergistically enhanced the cytotoxicity and cell growth inhibition of erlotinib in NSCLC cells; these effects were associated with Rad52 downregulation and p38 MAPK inactivation in H520 and H1703 cells. Conclusion: The results provide a rationale for combining 5-FU and erlotinib in lung cancer treatment.

CKAP2L, as an Independent Risk Factor, Closely Related to the Prognosis of Glioma.

Recent studies have found that cytoskeleton-associated protein 2 like (CKAP2L), an important oncogene, is involved in the biological behavior of many malignant tumors, but its function in the malignant course of glioma has not been confirmed. The main purpose of this study was to clarify the relationship between prognostic clinical characteristics of glioma patients and CKAP2L expression using data collected from the GEPIA, HPA, CGGA, TCGA, and GEO databases. CKAP2L expression was significantly increased in glioma. Further, Kaplan-Meier plots revealed that increased expression of CKAP2L was associated with shorter survival time of glioma patients in datasets retrieved from multiple databases. Cox regression analysis indicated that CKAP2L can serve as an independent risk factor but also has relatively reliable diagnostic value for the prognosis of glioma patients. The results of gene set enrichment analysis suggested that CKAP2L may play a regulatory role through the cell cycle, homologous recombination, and N-glycan biosynthesis cell signaling pathways. Several drugs with potential inhibitory effects on CKAP2L were identified in the CMap database that may have therapeutic effects on glioma. Finally, knockdown of CKAP2L inhibited the proliferation and invasion of cells by reducing the expression level of cell cycle-related proteins. This is the first study to demonstrate that high CKAP2L expression leads to poor prognosis in glioma patients, providing a novel target for diagnosis and treatment of glioma.