TrAEL-seq for WRN inhibitors
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1064407 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27536356.fastq.gz | Fastqsanger.gz | |||
| SRR27536357.fastq.gz | Fastqsanger.gz | |||
| SRR27536358.fastq.gz | Fastqsanger.gz | |||
| SRR27536359.fastq.gz | Fastqsanger.gz | |||
| SRR27536360.fastq.gz | Fastqsanger.gz |
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Project description:TrAEL-seq with WRN inhibitors in sensitive and resistant cells
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Project description:Cancers with microsatellite instability (MSI) depend on the WRN helicase enzyme to manage issues during DNA replication caused by long stretches of (TA) repeats in the DNA. Targeting WRN is a promising strategy for treating MSI cancers, and drugs that inhibit WRN are being developed. Through a process called fragment-based screening, we developed powerful and specific drugs that block WRN's action. These drugs effectively slowed down the growth of MSI cancer models in lab and animal studies by acting like WRN is absent, leading to DNA breaks at the long TA repeats and causing DNA damage. The development of these potent and specific drugs targeting WRN in MSI cancers proves that this approach can work and also helps us understand more about WRN's role in biology.
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Project description:Werner helicase inhibitors (WRNi) show promise for treating patients with microsatellite-unstable (MSI) tumors characterized by defective DNA mismatch repair. Multiple WRNi recently entered Phase 1 clinical trials. Here, we investigate the impact of cancer cell evolutionary adaptation on WRN pharmacological inhibition with implications for understanding drug selectivity and potential clinical resistance. Coupling genome-wide CRISPR screens with WRN gene knockout, no suppressors of WRN dependency were identified, underscoring WRN’s essential non-redundant function in MSI cells. Pharmacogenomic screens pinpointed modulators of WRNi sensitivity, including SMARCAL1, linking WRN-MSI synthetic lethality with chromatin remodelling and DNA repair pathways. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving spontaneous secondary resistance to multiple WRNi in vitro and in vivo. Specific resistance mutations preserve sensitivity to alternative WRNi, whereas others induce cross-resistance. Our work guides next-generation strategies for targeting MSI cancers, enabling cross-resistance studies to evaluate current and novel WRNi efficacy and informing future clinical trial design.
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Project description:Precise DNA replication is critical to the maintenance of genome stability, and the DNA replication machinery is a focal point of many current and upcoming chemotherapeutics. TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronised cells and does not require treatment with drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq including multiplexing of up to 6 samples that dramatically improves sample quality and throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples, and does not require cell synchronisation, drug treatment, labelling or sorting. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication initiation zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, which is dominated by rapid fork movement in long replication regions (>1Mb). Short and long replication regions are not intrinsically different, and instead replication forks accelerate across the first ~1 Mb of travel such that forks progress faster in the middle of regions lying between widely spaced initiation zones. We propose that this is a natural consequence of fewer replication forks being active later in S phase when these distal regions are replicated and there being less competition for replication factors.
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Project description:We have applied the TrAEL-seq method to profile DNA replication in wild type and Snf2h knockoutmouse embryonic stem cells.
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