Similar Datasets
Project description:Alteration of the gene encoding BRCA2, a protein involved in homologous recombination (HR), by missense or nonsense mutations results in sensitivity of a subset of breast and ovarian cancers to platinating agents and poly(ADP-ribose) polymerase (PARP) inhibitors. Conversely, resistance to these agents is often accompanied by secondary BRCA2 mutations that restore a functional protein. Here we characterized clones derived from the BRCA2-mutant ovarian cancer line PEO1 by selection with the PARP inhibitor veliparib. Colony forming assays demonstrated cross-resistance of the resulting clones to multiple PARP inhibitors and cisplatin. Despite the continued presence of a 4965C>A (p.Y1655X) BRCA2 nonsense mutation, HR was restored in the resistant cells. Immunoprecipitation followed by immunoblotting or mass spectrometry demonstrated expression of low levels of BRCA2 protein, including peptides distal to the premature termination codon. Reporter assays demonstrated readthrough selective for the UAG stop codon in the veliparib-resistant clones but not parental PEO1 cells; and mass spectrometry revealed insertion of tyrosine rather than termination in the readthrough product. Mass spectrometry and immunoblotting provided evidence for readthrough of UAGs in additional proteins in the resistant clones. BRCA2 gene interruption 3’ to the stop codon restored sensitivity of the resistant clones to PARP inhibitors and cisplatin, implicating the low level BRCA2 expression in resistance. Accordingly, the present results identify low level readthrough of nonsense codons as a potential mechanism of drug resistance that might need to be considered when assessing the impact of mutations on the biology of cancers.
2025-10-27 | PXD037989 | Pride
Project description:Deleterious alterations of the gene encoding BRCA2, a protein involved in homologous recombination (HR), contribute to the pathogenesis of a subset of ovarian, breast, prostate and pancreatic cancers as well as their increased sensitivity to platinating agents and poly(ADP-ribose) polymerase inhibitors (PARPis). While secondary mutations that restore a functional BRCA2 protein are detected in a fraction of cases that become drug resistant, the causes of resistance in other cases are poorly understood. Here we characterized clones derived from the BRCA2-mutant ovarian cancer line PEO1 by PARPi selection. Colony forming assays demonstrated cross-resistance of these clones to multiple PARPis, cisplatin and doxorubicin. Despite persistence of the signature 4965C>G (p.Y1655X) BRCA2 nonsense mutation, HR was restored in the resistant cells. Immunoprecipitation followed by immunoblotting or mass spectrometry demonstrated expression of low levels of BRCA2 protein, including peptides distal to the premature termination codon. Reporter assays demonstrated readthrough selective for the UAG stop codon in the resistant clones but not parental PEO1 cells. Mass spectrometry and immunoblotting provided evidence for readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. BRCA2 gene interruption either 5’ or 3’ to the stop codon restored sensitivity of the resistant clones to PARPis and cisplatin, implicating the low level BRCA2 expression in resistance. Accordingly, the present results identify low level readthrough of nonsense codons as a potential mechanism of drug resistance that might need to be considered when assessing the impact of BRCA2 nonsense mutations.
2024-09-01 | GSE243142 | GEO
Project description:Deleterious alterations of the gene encoding BRCA2, a protein involved in homologous recombination (HR), contribute to the pathogenesis of a subset of ovarian, breast, prostate and pancreatic cancers as well as their increased sensitivity to platinating agents and poly(ADP-ribose) polymerase inhibitors (PARPis). While secondary mutations that restore a functional BRCA2 protein are detected in a fraction of cases that become drug resistant, the causes of resistance in other cases are poorly understood. Here we characterized clones derived from the BRCA2-mutant ovarian cancer line PEO1 by PARPi selection. Colony forming assays demonstrated cross-resistance of these clones to multiple PARPis, cisplatin and doxorubicin. Despite persistence of the signature 4965C>G (p.Y1655X) BRCA2 nonsense mutation, HR was restored in the resistant cells. Immunoprecipitation followed by immunoblotting or mass spectrometry demonstrated expression of low levels of BRCA2 protein, including peptides distal to the premature termination codon. Reporter assays demonstrated readthrough selective for the UAG stop codon in the resistant clones but not parental PEO1 cells. Mass spectrometry and immunoblotting provided evidence for readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. BRCA2 gene interruption either 5’ or 3’ to the stop codon restored sensitivity of the resistant clones to PARPis and cisplatin, implicating the low level BRCA2 expression in resistance. Accordingly, the present results identify low level readthrough of nonsense codons as a potential mechanism of drug resistance that might need to be considered when assessing the impact of BRCA2 nonsense mutations.
2025-10-22 | GSE243141 | GEO
Project description:Codon Specific Readthrough as a Mechanism of BRCA2 Restoration in Acquired PARP Inhibitor and Chemotherapy Resistance
| PRJNA1016421 | ENA
Project description:Codon Specific Readthrough as a Mechanism of BRCA2 Restoration in Acquired PARP Inhibitor and Chemotherapy Resistance [WES]
| PRJNA1016426 | ENA
Project description:Codon Specific Readthrough as a Mechanism of BRCA2 Restoration in Acquired PARP Inhibitor and Chemotherapy Resistance [RNA-seq]
| PRJNA1016425 | ENA
Project description:Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a major advance in ovarian cancer, now as a treatment and as a maintenance therapy in the upfront and recurrent settings. However, patients often develop resistance to PARPis, underlining the importance of dissecting resistance mechanisms. Here, we report different dosing/timing schemes of PARPi treatment in BRCA2-mutant PEO1 cells, resulting in the simultaneous development of distinct resistance mechanisms. PARPi-resistant variants PEO1/OlaJR, established by higher initial doses and short-term PARPi treatment, develops PARPi resistance by rapidly restoring functional BRCA2 and promoting drug efflux activity. In contrast, PEO1/OlaR, developed by lower initial doses with long-term PARPi exposure, shows no regained BRCA2 function but a mesenchymal-like phenotype with greater invasion ability, and exhibits activated ATR/CHK1 and suppressed EZH2/MUS81 signaling cascades to regain HR repair and fork stabilization, respectively. Our study suggests that PARPi resistance mechanisms can be governed by treatment strategies and have a molecular basis on BRCA2 functionality. Further, we define different mechanisms that may serve as useful biomarkers to assess subsequent treatment strategies in PARPi-resistant ovarian cancer.
| S-EPMC9669252 | biostudies-literature
Project description:Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.
| S-EPMC2913123 | biostudies-literature
Project description:Breast cancer gene 2 (BRCA2) deleterious mutations confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition due to its critical role in DNA repair. PARP inhibitor Olaparib is now approved and several other PARP inhibitors are now in different stages of clinical trials. Development of resistance to PARP inhibitors limits their clinical utility. The mechanism of resistance remains not fully understood. Here we show that amplification of mutant BRCA2 confers PARP inhibitor resistance. The amplification of mutant BRCA2 gene copies correlates with an increase in mutant BRCA2 expression and an increase in the levels of its interacting proteins PALB2 and RAD51. In addition, homologous recombination mediated DNA repair is rescued in these cells as evidenced by the formation of RAD51 focus formation. The overexpressed mutant BRCA2 is essential for the observed PARP inhibitor resistance because knockdown of its expression is sufficient to re-sensitize these cells to PARP inhibition. Collectively, our results indicate a new mechanism of resistance to PARP inhibitor in BRCA2 mutant cancer cells
2017-09-02 | GSE86394 | GEO
Project description:The advanced development of synthetic lethality has opened the doors for specific anti-cancer medications of personalized medicine and efficient therapies against cancers. One of the most popular approaches being investigated is targeting DNA repair pathways as the implementation of the PARP inhibitor (PARPi) into individual or combinational therapeutic schemes. Such treatment has been effectively employed against homologous recombination-defective solid tumors as well as hematopoietic malignancies. However, the resistance to PARPi has been observed in both preclinical research and clinical treatment. Therefore, elucidating the mechanisms responsible for the resistance to PARPi is pivotal for the further success of this intervention. Apart from mechanisms of acquired resistance, the bone marrow microenvironment provides a pre-existing mechanism to induce the inefficiency of PARPi in leukemic cells. Here, we describe the pre-existing and acquired mechanisms of the resistance to PARPi-induced synthetic lethality. We also discuss the potential rationales for developing effective therapies to prevent/repress the PARPi resistance in cancer cells.
| S-EPMC9741207 | biostudies-literature