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Transcription-Replication Conflicts drive the Synthetic Lethality of PARP Inhibitors with BRCA2-Deficiency

ABSTRACT: To examine replication fork progression firing upon TIMELESS/TIPIN knockdown, EdUseq was performed in HeLa cells transfected with siCTRL, siTIMELESS or siTIPIN, synchronised with Thymidine and then released for 120 min in S phase, with the last 30 minutes being labeled with EdU.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220223 | GEO | 2024/04/12

REPOSITORIES: GEO

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The mismatch recognition protein MutSalpha promotes nascent strand degradation at stalled replication forks

Project description:Regular iPOND: 707789: C3-4 EdU, 707790: C3-4 Thymidine chase, 707791: A3-6 EdU, 707792: A3-6 Thymidine chase. SILAC iPOND: 1002641. Regular iPOND: WT MEF cell line (C3-4) and P286R MEF cells (A3-6) were labeled with EdU with (C3-4/A3-6 Thymidine chase) or without thymidine chase (C3-4/A3-6 EdU). Click reaction was performed to link a biotin to the EdU followed by pull down with streptavidin beads. SILAC iPOND: P286R MEF cells (abundance 1002641 Light) were cultured in "light" medium containing [12C14N]-L-lysine and [12C14N]-L-arginine, and WT MEF cells (Abundance 1002641 Heavy) were cultured in "heavy" medium containing [13C15N]-L-lysine [13C15N]-L-arginine. After more than 99% cells were labeled with isotopes, equal amount of WT and P286R cells were combined followed by EdU labeling and click reaction. Proteins captured were dissolved on SDS/PAGE and subjected to MS analysis.

| MSV000090330 | MassIVE

Transcription-Replication Conflicts drive the Synthetic Lethality of PARP Inhibitors with BRCA2-Deficiency

Project description:Transcription-Replication Conflicts drive the Synthetic Lethality of PARP Inhibitors with BRCA2-Deficiency

| PRJNA909256 | ENA

Nucleotide Excision Repair Removes Thymidine Analog 5-Ethynyl-2’-deoxyuridine From the Human Genome

Project description:5-Ethynyl-2’-deoxyuridine (EdU) is one of the several thymidine analogs that are used in labeling DNA in cells and in DNA combing (DNA fiber analysis) to study DNA replication1-5. We wished to use these analogs to investigate the repair complex (DNA-protein) of nucleotide excision repair. UV irradiated human cell lines were incubated with EdU, BrdU, IdU, CldU, F-ara-EdU or AmdU to incorporate these analogs into the 26-27 nucleotide long excision repair products. Unexpectedly, we found that EdU even in the absence of DNA damage was released in the form of 26-27nt-long oligomers. We conclude that EdU is incorporated into replicating DNA and is recognized as damage by the nucleotide excision repair system and removed from the genome followed by repair synthesis and re-incorporation into DNA, thus creating futile cycle that leads to cell death. This property of EdU, which was not seen with any of the other thymidine analogs, may be taken advantage of in treating cancer, in particular brain cancers because EdU crosses blood-brain barrier.

2022-08-02 | GSE202784 | GEO

A high-resolution, nanopore-based artificial intelligence assay for DNA replication stress in human cancer cells

Project description:Human cancer cell lines were pulsed with thymidine analogues EdU and BrdU, sequenced on the Oxford Nanopore platform, and analysed with our DNAscent software to measure DNA replication stress.

2024-04-01 | GSE216926 | GEO

Attenuation of SRC Kinase Activity Augments PARP Inhibitor-mediated Synthetic Lethality in <i>BRCA2</i>-altered Prostate Tumors.

Project description:PurposeAlterations in DNA damage repair (DDR) pathway genes occur in 20%-25% of men with metastatic castration-resistant prostate cancer (mCRPC). Although PARP inhibitors (PARPis) have been shown to benefit men with mCRPC harboring DDR defects due to mutations in BRCA1/2 and ATM, additional treatments are necessary because the effects are not durable.Experimental designWe performed transcriptomic analysis of publicly available mCRPC cases, comparing BRCA2 null with BRCA2 wild-type. We generated BRCA2-null prostate cancer cells using CRISPR/Cas9 and treated these cells with PARPis and SRC inhibitors. We also assessed the antiproliferative effects of combination treatment in 3D prostate cancer organoids.ResultsWe observed significant enrichment of the SRC signaling pathway in BRCA2-altered mCRPC. BRCA2-null prostate cancer cell lines had increased SRC phosphorylation and higher sensitivity to SRC inhibitors (e.g., dasatinib, bosutinib, and saracatinib) relative to wild-type cells. Combination treatment with PARPis and SRC inhibitors was antiproliferative and had a synergistic effect in BRCA2-null prostate cancer cells, mCRPC organoids, and Trp53/Rb1-null prostate cancer cells. Inhibition of SRC signaling by dasatinib augmented DNA damage in BRCA2-null prostate cancer cells. Moreover, SRC knockdown increased PARPi sensitivity in BRCA2-null prostate cancer cells.ConclusionsThis work suggests that SRC activation may be a potential mechanism of PARPi resistance and that treatment with SRC inhibitors may overcome this resistance. Our preclinical study demonstrates that combining PARPis and SRC inhibitors may be a promising therapeutic strategy for patients with BRCA2-null mCRPC.

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Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

Project description:The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

2020-10-08 | GSE144321 | GEO

Transcription-replication conflicts underlie sensitivity to PARP inhibitors.

Project description:An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1-6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

| S-EPMC11006605 | biostudies-literature

PARP inhibitors: Synthetic lethality in the clinic.

Project description:PARP inhibitors (PARPi), a cancer therapy targeting poly(ADP-ribose) polymerase, are the first clinically approved drugs designed to exploit synthetic lethality, a genetic concept proposed nearly a century ago. Tumors arising in patients who carry germline mutations in either BRCA1 or BRCA2 are sensitive to PARPi because they have a specific type of DNA repair defect. PARPi also show promising activity in more common cancers that share this repair defect. However, as with other targeted therapies, resistance to PARPi arises in advanced disease. In addition, determining the optimal use of PARPi within drug combination approaches has been challenging. Nevertheless, the preclinical discovery of PARPi synthetic lethality and the route to clinical approval provide interesting lessons for the development of other therapies. Here, we discuss current knowledge of PARP inhibitors and potential ways to maximize their clinical effectiveness.

| S-EPMC6175050 | biostudies-literature

CDK12 prevents MYC-induced transcription-replication conflicts [EdU-seq]

Project description:CDK12 prevents MYC-induced transcription-replication conflicts [EdU-seq]

| PRJNA991567 | ENA

Enhancement of synthetic lethality via combinations of ABT-888, a PARP inhibitor, and carboplatin in vitro and in vivo using BRCA1 and BRCA2 isogenic models.

Project description:Individuals with an inherited BRCA1 or BRCA2 mutation have an elevated risk of developing breast cancer. The resulting tumors typically lack homologous recombination repair as do a subset of sporadic tumors with acquired BRCA deficiency. Clinical responses to monotherapy with platinum drugs or poly PARP inhibitors (PARPi) have been shown for BRCA-associated cancers. However, there are limited data on combination therapy with PARPi and platinum drugs, the mechanism of action of this combination, and the role of BRCA1 or BRCA2 in chemosensitivity. We compared the efficacy of ABT-888 (a PARPi) with that of cisplatin or carboplatin (platinum drugs) alone or in combinations by examining the survival of treated Brca-proficient and -deficient mouse embryonic stem cells. In addition, drug-induced growth inhibition of a BRCA1 and a BRCA2 null cell line were compared with their isogenic BRCA-complemented lines. Although each monotherapy killed or inhibited proliferation of Brca/BRCA-deficient cells, an enhanced effect was observed after treatment with ABT-888 in combination with carboplatin. Moreover, the ABT-888/carboplatin combination delayed tumor growth in Brca2 xenografts. The drugs caused DNA damage and apoptosis. Along with greater PARP activity in Brca/BRCA-deficient cells, these effects correlated with increased chemosensitivity. Our data suggest that ABT-888 and carboplatin combination treatment will be more successful than monotherapy in addressing many BRCA-associated cancers. A randomized phase II trial has recently been initiated to test this hypothesis to assist in the discovery of more effective therapies for patients with BRCA.

| S-EPMC3551628 | biostudies-literature

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