Biology subjects

Cowley, K.

Publications and source records attributed to Cowley, K..

4 recordsLinked to original sources

bioRxiv2026

Cell cycle dependent regulation of mRNA export in response to replication stress

Nuclear export of mRNA is extensively coupled to transcription and processing of mRNA. How mRNA export is mechanistically regulated remains poorly understood. Here, we uncover a physical checkpoint at nuclear speckles that regulates mRNA export during DNA replication in S-phase of the cell cycle. Unbiased screening approaches identify WEE1 and CHK1 as novel mRNA export regulators. mRNA export complexes are recruited to sites of replication stress in S-phase. WEE1 inhibition prematurely activates CDK1 and PLK1, leading to accumulation of R-loop associated mRNA in large nuclear speckles, with late markers of replication stress present around their periphery. This recruitment is dependent on CDK1 activity. Phosphorylation of ALYREF by CDK1 and PLK1 regulates nuclear speckle accumulation of mRNA following replication stress. mRNA export factors including ALYREF are subsequently mis-localised from these speckles, preventing nuclear export of R-loop associated mRNA. Thus, WEE1, CDK1 and PLK1 enforce a cell cycle regulated checkpoint at nuclear speckles that serves to protect the cell from major sources of genome instability by ensuring that R-loop associated mature mRNA is not exported to the cytoplasm.

molecular biology↗

bioRxiv2026

Fanconi Anaemia E3 Ligase complex activity is regulated by a druggable metabolite binding site in FANCX

The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro. Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1-deficient cells in vitro. These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

cancer biology↗

bioRxiv2026

Targeting DNA Methylation Reactivates Type I Interferon Signalling in Bone-Metastatic Breast Cancer

Bone metastasis remains a major clinical challenge in advanced breast cancer. Downregulation of type I interferon signalling, a critical immunomodulatory pathway in anti-cancer immunity and disease progression, is a defining feature of this process. Here, we utilised an IFN-reporter system to perform unbiased epigenetic compound screens to identify agents that could restore type I IFN signalling. This screen identified Decitabine, a DNA hypomethylating agent, that enhanced tumor immunogenicity across a broad range of mouse and human breast cancer cell lines, including bone-derived lines. Mechanistically, suppression of interferon-stimulated genes is highly correlated with elevated DNMT1 expression in bone metastasis compared with primary tumor, in both mouse models and matched human samples. Decitabine treatment was sufficient to reactivate interferon stimulated genes in bone-derived 4T1.2 cell lines via hypomethylation of type I interferon pathway gene promoter regions. Correspondingly, in the syngeneic 4T1.2 metastasis mouse model, Decitabine treatment conferred a survival benefit and reduced metastatic potential, particularly in bone. Our findings reveal DNA methylation as a key regulator of the transcriptional programs underlying bone metastasis, providing mechanistic insight into how Decitabine reactivates type I interferon signalling and reduces metastatic potential, highlighting epigenetic reprogramming as a promising approach for targeting metastatic breast cancer. STATEMENT OF SIGNIFICANCEBone metastasis remains a major clinical challenge and a key mechanism of progression to bone is the suppression of tumor-inherent type I interferon signalling. We identified Decitabine, a DNA hypomethylating agent, as a promising therapeutic agent to enhance tumor immunogenicity across a broad range of breast cancer cell lines, including bone metastasis-derived lines. Our findings support DNA methylation as a key regulator of transcriptional programs associated with bone metastatic progression, and provide mechanistic insight into how Decitabine reactivates type I interferon signalling and reduces metastatic potential in vivo. These results highlight epigenetic reprogramming as a promising approach for targeting metastatic breast cancer.

Cancer Biology↗

bioRxiv2021

Targeting of TP53-independent cell cycle checkpoints overcomes FOLFOX resistance in Metastatic Colorectal Cancer

Patients with colorectal cancer (CRC) frequently develop liver metastases during the course of their disease. A substantial proportion of them receive neoadjuvant FOLFOX (5-Fluorouracil, Oxaliplatin, Leucovorin) prior to surgery in an attempt to enable successful surgical removal of their metastases and to reduce the risk of recurrence. Yet, the majority of patients progress during treatment or recur following surgery, and molecular mechanisms that contribute to FOLFOX resistance remain poorly understood. Here, using a combination of phenotypic, transcriptomic and genomic analyses of both tumor samples derived from patients with metastatic CRC and matching patient-derived tumor organoids (PDTOs), we characterize a novel FOLFOX resistance mechanism and identify inhibitors that target this mechanism to resensitize metastatic organoids to FOLFOX. Resistant PDTOs, identified after in vitro exposure to FOLFOX, exhibited elevated expression of E2F pathway, S phase, G2/M and spindle assembly checkpoints (SAC) genes. Similar molecular features were detected in CRLM from patients with progressive disease while under neoadjuvant FOLFOX treatment, highlighting the relevance of this finding. FOLFOX resistant PDTOs displayed inactivating mutations of TP53 and exhibited transcriptional features of P53 pathway downregulation. We found that they accumulated in early S-phase and underwent significant DNA damage during FOLFOX exposure, thereafter arresting in G2/M while they repaired their DNA after FOLFOX withdrawal. In parallel, results of a large kinase inhibitor screen indicated that drugs targeting regulators of the DNA damage response, G2M checkpoint and SAC had cytotoxic effects on PDTOs generated from patients whose disease progressed during treatment with FOLFOX. Corroborating this finding, CHK1 and WEE1 inhibitors were found to synergize with FOLFOX and sensitize previously resistant PDTOs. Additionally, targeting the SAC master regulator MPS1 using empesertib after exposure to FOLFOX, when cells accumulate in G2M, was also very effective to kill FOLFOX-resistant PDTOs. Our results indicate that targeted and timely inhibition of specific cell cycle checkpoints shows great potential to improve response rates to FOLFOX in patients with metastatic CRC, for whom therapeutic alternatives remain extremely limited.

cancer biology↗