From desert flora to cancer therapy: systematic exploration of multi-pathway mechanisms using network pharmacology and molecular modeling approaches.

Ovarian cancer, often labeled a "silent killer," remains one of the most compelling and challenging areas of cancer research. In 2019 alone, a staggering 222,240 new cases of ovarian cancer were reported, with nearly 14,170 lives tragically lost to this relentless disease. The absence of effective diagnostic methods, increased resistance to chemotherapy, and the heterogeneous nature of ovarian cancer collectively contribute to the unfavorable prognosis observed in the majority of cases. Thus, there is a pressing need to explore therapeutic interventions that offer superior efficacy and safety, thereby enhancing the survival prospects for ovarian cancer patients. Recognizing this potential, our research synergizes bioinformatics with a network pharmacology approach to investigate the underlying molecular interactions of Saudi Arabian flora (Onopordum heteracanthum, Acacia ehrenbergiana, Osteospermum vaillantii, Cyperus rotundus, Carissa carandas, Carissa spinarum, and Camellia sinensis) in ovarian cancer treatment. At first, phytoconstituents of indigenous flora and their associated gene targets, particularly those pertinent to ovarian cancer, were obtained from open-access databases. Later, the shared targets of plants and diseases were compared to identify common targets. A protein-protein interaction (PPI) network of predicted targets was then constructed for the identification of key genes having the highest degree of connectivity among networks. Following that, a compound-target protein-pathway network was constructed, which uncovered that, namely, hispidulin, stigmasterol, ascorbic acid, octopamine, cyperene, kaempferol, pungenin, citric acid, d-tartaric acid, beta-sitosterol, (-)-epicatechin gallate, and (+)-catechin demonstrably influence cell proliferation and growth by impacting the AKT1 and VEGFA proteins. Molecular docking, complemented by a 20-ns molecular dynamic (MD) simulation, was used, and the binding affinity of the compound was further validated. Molecular docking, complemented by a 20-ns MD simulation, confirmed the binding affinity of these compounds. Specifically, for AKT1, ascorbic acid showed a docking score of -11.1227 kcal/mol, interacting with residues Ser A:240, Leu A:239, Arg A:243, Arg C:2, and Glu A:341. For VEGFA, hispidulin exhibited a docking score of -17.3714 kcal/mol, interacting with Asn A:158, Val A:190, Gln B:160, Ser A:179, and Ser B:176. To sum up, both a theoretical and empirical framework were established by this study, directing more comprehensive research and laying out a roadmap for the potential utilization of active compounds in the formulation of anti-cancer treatments.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Unravelling the clinicopathological and functional significance of replication protein A (RPA) heterotrimeric complex in breast cancers.

Replication Protein A (RPA), a heterotrimeric complex consisting of RPA1, 2, and 3 subunits, is a single-stranded DNA (ssDNA)-binding protein that is critically involved in replication, checkpoint regulation and DNA repair. Here we have evaluated RPA in 776 pure ductal carcinomas in situ (DCIS), 239 DCIS that co-exist with invasive breast cancer (IBC), 50 normal breast tissue and 4221 IBC. Transcriptomic [METABRIC cohort (n = 1980)] and genomic [TCGA cohort (n = 1090)] evaluations were completed. Preclinically, RPA deficient cells were tested for cisplatin sensitivity and Olaparib induced synthetic lethality. Low RPA linked to aggressive DCIS, aggressive IBC, and shorter survival outcomes. At the transcriptomic level, low RPA tumours overexpress pseudogene/lncRNA as well as genes involved in chemical carcinogenesis, and drug metabolism. Low RPA remains linked with poor outcome. RPA deficient cells are sensitive to cisplatin and Olaparib induced synthetic lethality. We conclude that RPA directed precision oncology strategy is feasible in breast cancers.

Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers.

Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n = 331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p = 0.002). In the ovarian cancer genome atlas (TCGA) cohort (n = 498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p < 0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n = 1259), Mre11 overexpression was associated with poor PFS (p = 0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.

Untangling the clinicopathological significance of MRE11-RAD50-NBS1 complex in sporadic breast cancers.

The MRE11-RAD50-NBS1 (MRN) complex is critical for genomic stability. Although germline mutations in MRN may increase breast cancer susceptibility, such mutations are extremely rare. Here, we have conducted a comprehensive clinicopathological study of MRN in sporadic breast cancers. We have protein expression profiled for MRN and a panel of DNA repair factors involved in double-strand break repair (BRCA1, BRCA2, ATM, CHK2, ATR, Chk1, pChk1, RAD51, γH2AX, RPA1, RPA2, DNA-PKcs), RECQ DNA helicases (BLM, WRN, RECQ1, RECQL4, RECQ5), nucleotide excision repair (ERCC1) and base excision repair (SMUG1, APE1, FEN1, PARP1, XRCC1, Pol ß) in 1650 clinical breast cancers. The prognostic significance of MRE11, RAD50 and NBS1 transcripts and their microRNA regulators (hsa-miR-494 and hsa-miR-99b) were evaluated in large clinical datasets. Expression of MRN components was analysed in The Cancer Genome Atlas breast cancer cohort. We show that low nuclear MRN is linked to aggressive histopathological phenotypes such as high tumour grade, high mitotic index, oestrogen receptor- and high-risk Nottingham Prognostic Index. In univariate analysis, low nuclear MRE11 and low nuclear RAD50 were associated with poor survival. In multivariate analysis, low nuclear RAD50 remained independently linked with adverse clinical outcomes. Low RAD50 transcripts were also linked with reduced survival. In contrast, overexpression of hsa-miR-494 and hsa-miR-99b microRNAs was associated with poor survival. We observed large-scale genome-wide alterations in MRN-deficient tumours contributing to aggressive behaviour. We conclude that MRN status may be a useful tool to stratify tumours for precision medicine strategies.

Discovery of Novel Inhibitors From Medicinal Plants for V-Domain Ig Suppressor of T-Cell Activation.

V-domain Ig suppressor of T cell activation (VISTA) is an immune checkpoint and is a type I transmembrane protein. VISTA is linked to immunotherapy resistance, and it is a potential immune therapeutic target, especially for triple-negative breast cancer. It expresses at a high concentration in regulatory T cells and myeloid-derived suppressor cells, and its functional blockade is found to delay tumor growth. A useful medicinal plant database for drug designing (MPD3), which is a collection of phytochemicals from diverse plant families, was employed in virtual screening against VISTA to prioritize natural inhibitors against VISTA. Three compounds, Paratocarpin K (PubChem ID: 14187087), 3-(1H-Indol-3-yl)-2-(trimethylazaniumyl)propanoate (PubChem ID: 3861164), and 2-[(5-Benzyl-4-ethyl-1,2,4-triazol-3-yl)sulfanylmethyl]-5-methyl-1,3,4-oxadiazole (PubChem ID: 6494266), having binding energies stronger than -6 kcal/mol were found to have two common hydrogen bond interactions with VISTA active site residues: Arg54 and Arg127. The dynamics of the compound-VISTA complexes were further explored to infer binding stability of the systems. Results revealed that the compound 14187087 and 6494266 systems are highly stable with an average RMSD of 1.31 Å. Further affirmation on the results was achieved by running MM-GBSA on the MD simulation trajectories, which re-ranked 14187087 as the top-binder with a net binding energy value of -33.33 kcal/mol. In conclusion, the present study successfully predicted natural compounds that have the potential to block the function of VISTA and therefore can be utilized further in experimental studies to validate their real anti-VISTA activity.

Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.

Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown. Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells. Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids. Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.

Mutational Landscape of Pirin and Elucidation of the Impact of Most Detrimental Missense Variants That Accelerate the Breast Cancer Pathways: A Computational Modelling Study.

Pirin (PIR) protein is highly conserved in both prokaryotic and eukaryotic organisms. Recently, it has been identified that PIR positively regulates breast cancer cell proliferation, xenograft tumor formation, and metastasis, through an enforced transition of G1/S phase of the cell cycle by upregulation of E2F1 expression at the transcriptional level. Keeping in view the importance of PIR in many crucial cellular processes in humans, we used a variety of computational tools to identify non-synonymous single-nucleotide polymorphisms (SNPs) in the PIR gene that are highly deleterious for the structure and function of PIR protein. Out of 173 SNPs identified in the protein, 119 are non-synonymous, and by consensus, 24 mutations were confirmed to be deleterious in nature. Mutations such as V257A, I28T, and I264S were unveiled as highly destabilizing due to a significant stability fold change on the protein structure. This observation was further established through molecular dynamics (MD) simulation that demonstrated the role of the mutation in protein structure destability and affecting its internal dynamics. The findings of this study are believed to open doors to investigate the biological relevance of the mutations and drugability potential of the protein.

FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers.

FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin ß. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLß and XRCC1. FEN1i treatment was selectively toxic to POLß deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.

Molecular disruption of DNA polymerase ß for platinum sensitisation and synthetic lethality in epithelial ovarian cancers.

Targeting PARP1 [Poly(ADP-Ribose) Polymerase 1] for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase ß (Polß), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polß deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polß depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polß small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG [Poly(ADP-Ribose) Glycohydrolase] inhibitor (PDD00017273) [but not PARP1 inhibitor (Olaparib)] was synthetically lethal in polß deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD+) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polß-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polß targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.

Clinicopathological and Functional Evaluation Reveal NBS1 as a Predictor of Platinum Resistance in Epithelial Ovarian Cancers.

Platinum resistance seriously impacts on the survival outcomes of patients with ovarian cancers. Platinum-induced DNA damage is processed through DNA repair. NBS1 is a key DNA repair protein. Here, we evaluated the role of NBS1 in ovarian cancers. NBS1 expression was investigated in clinical cohorts (protein level (n = 331) and at the transcriptomic level (n = 1259)). Pre-clinically, sub-cellular localization of NBS1 at baseline and following cisplatin therapy was tested in platinum resistant (A2780cis, PEO4) and sensitive (A2780, PEO1) ovarian cancer cells. NBS1 was depleted and cisplatin sensitivity was investigated in A2780cis and PEO4 cells. Nuclear NBS1 overexpression was associated with platinum resistance (p = 0.0001). In univariate and multivariate analysis, nuclear NBS1 overexpression was associated with progression free survival (PFS) (p-values = 0.003 and 0.017, respectively) and overall survival (OS) (p-values = 0.035 and 0.009, respectively). NBS1 mRNA overexpression was linked with poor PFS (p = 0.011). Pre-clinically, following cisplatin treatment, we observed nuclear localization of NBS1 in A2780cis and PEO4 compared to A2780 and PEO1 cells. NBS1 depletion increased cisplatin cytotoxicity, which was associated with accumulation of double strand breaks (DSBs), S-phase cell cycle arrest, and increased apoptosis. NBS1 is a predictor of platinum sensitivity and could aid stratification of ovarian cancer therapy.

XRCC1 deficient triple negative breast cancers are sensitive to ATR, ATM and Wee1 inhibitor either alone or in combination with olaparib.

BACKGROUND: PARP inhibitor (PARPi) monotherapy is a new strategy in BRCA germ-line deficient triple negative breast cancer (TNBC). However, not all patients respond, and the development of resistance limits the use of PARPi monotherapy. Therefore, the development of alternative synthetic lethality strategy, including in sporadic TNBC, is a priority. XRCC1, a key player in base excision repair, single strand break repair, nucleotide excision repair and alternative non-homologous end joining, interacts with PARP1 and coordinates DNA repair. ATR, ATM and Wee1 have essential roles in DNA repair and cell cycle regulation. METHODS: Highly selective inhibitors of ATR (AZD6738), ATM (AZ31) and Wee1 (AZD1775) either alone or in combination with olaparib were tested for synthetic lethality in XRCC1 deficient TNBC or HeLa cells. Clinicopathological significance of ATR, ATM or Wee1 co-expression in XRCC1 proficient or deficient tumours was evaluated in a large cohort of 1650 human breast cancers. RESULTS: ATR (AZD6738), ATM (AZ31) or Wee1 (AZD1775) monotherapy was selectively toxic in XRCC1 deficient cells. Selective synergistic toxicity was evident when olaparib was combined with AZD6738, AZ31 or AZD1775. The most potent synergistic interaction was evident with the AZD6738 and olaparib combination therapy. In clinical cohorts, ATR, ATM or Wee1 overexpression in XRCC1 deficient breast cancer was associated with poor outcomes. CONCLUSION: XRCC1 stratified DNA repair targeted combinatorial approach is feasible and warrants further clinical evaluation in breast cancer.

RAD50 deficiency is a predictor of platinum sensitivity in sporadic epithelial ovarian cancers.

Intrinsic or acquired resistance seriously limits the use of platinating agents in advanced epithelial ovarian cancers. Increased DNA repair capacity is a key route to platinum resistance. RAD50 is a critical component of the MRN complex, a 'first responder' to DNA damage and essential for the repair of DSBs and stalled replication forks. We hypothesised a role for RAD50 in ovarian cancer pathogenesis and therapeutics. Clinicopathological significance of RAD50 expression was evaluated in clinical cohorts of ovarian cancer at the protein level (n = 331) and at the transcriptomic level (n = 1259). Sub-cellular localization of RAD50 at baseline and following cisplatin therapy was tested in platinum resistant (A2780cis, PEO4) and sensitive (A2780, PEO1) ovarian cancer cells. RAD50 was depleted and cisplatin sensitivity was investigated in A2780cis and PEO4 cells. RAD50 deficiency was associated with better progression free survival (PFS) at the protein (p = 0.006) and transcriptomic level (p < 0.001). Basal level of RAD50 was higher in platinum resistant cells. Following cisplatin treatment, increased nuclear localization of RAD50 was evident in A2780cis and PEO4 compared to A2780 and PEO1 cells. RAD50 depletion using siRNAs in A2780cis and PEO4 cells increased cisplatin cytotoxicity, which was associated with accumulation of DSBs, S-phase cell cycle arrest and increased apoptosis. We provide evidence that RAD50 deficiency is a predictor of platinum sensitivity. RAD50 expression-based stratification and personalization could be viable clinical strategy in ovarian cancers.

PARP1 blockade is synthetically lethal in XRCC1 deficient sporadic epithelial ovarian cancers.

PARP1 inhibitor (Niraparib, Olaparib, Rucaparib) maintenance therapy improves progression-free survival in platinum sensitive sporadic epithelial ovarian cancers. However, biomarkers of response to PARPi therapy is yet to be clearly defined. XRCC1, a scaffolding protein, interacts with PARP1 during BER and SSBR. In a large clinical cohort of 525 sporadic ovarian cancers, high XRCC1 or high PARP1 protein levels was not only associated with aggressive phenotypes but was also significantly linked with poor progression-free survival (p = 0.048 & p = 0.001 respectively) and poor ovarian cancer-specific survival (p = 0.020 & p = 0.008 respectively). Pre-clinically, Olaparib and Talazoparib therapy were selectively toxic in XRCC1 deficient or knock-out platinum sensitive ovarian cancer cells in 2D and 3D models. Increased sensitivity was associated with DNA double-strand break accumulation, cell cycle arrest and apoptotic cell accumulation. We conclude that XRCC1 deficiency predicts sensitivity to PARP inhibitor therapy. PARP1 targeting is a promising new approach in XRCC1 deficient ovarian cancers.

ERCC1-XPF deficiency is a predictor of olaparib induced synthetic lethality and platinum sensitivity in epithelial ovarian cancers.

PURPOSE: PARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers. METHODS: ERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality. RESULTS: Low ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis. CONCLUSION: The data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.