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FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress

ABSTRACT: Oncogene-induced replication stress constitutes an early obstacle for pre-cancerous cells to overcome to progress towards malignancy. Fanconi anaemia signalling represents a major genomic maintenance pathway that is activated in response to replication stress, impinging on stalled replication fork stability and recovery. Here, we report that upon replication stress, phosphorylation of the FANCD2 N-terminus by CHK1 triggers FBXL12-dependent proteasomal degradation of FANCD2, facilitating clearance of FANCD2 at stalled replication forks. This mechanism is required to promote efficient and faithful DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Notably, reconstitution of FANCD2 with mutations in the N-terminal phosphodegron fail to re-establish fork progression in FANCD2-deficient human fibroblasts in response to replication stress. In the absence of FBXL12, FANCD2 becomes trapped on chromatin leading to replication stress, excessive DNA damage, and cell death. In human cancers, FBXL12, CYCLIN E, and Fanconi anaemia signalling are positively correlated and upregulation or amplification of FBXL12 is linked to reduced survival in patients with high CYCLIN E expressing breast tumours. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitised breast cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability of CYCLIN E-overexpressing cancer cells and may represent a novel target for cancer therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE232558 | GEO | 2023/08/16

REPOSITORIES: GEO

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Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

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FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress

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