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

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. Comparative gene expression profiling analysis of RNA-seq data obtained using MDA-MB-231 basal-like breast cancer cells. Triplicate samples from FBXL12-WT (transduced with scrambled control gRNA) reference cells and two FBXL12-KO clones were profiled.

致癌基因诱导的复制应激（oncogene-induced replication stress）是癌前细胞向恶性表型演进过程中必须克服的早期障碍。范可尼贫血信号通路（Fanconi anaemia signalling）是一类响应复制应激而激活的核心基因组维持通路，可调控停滞复制叉的稳定性与修复恢复过程。
在此，本研究报道，在复制应激状态下，CHK1对FANCD2蛋白N端的磷酸化修饰，会触发FBXL12介导的蛋白酶体降解途径，促使FANCD2从停滞复制叉处被清除。该机制对于细胞在细胞周期蛋白E（CYCLIN E）及药物诱导的复制应激条件下，实现高效且保真性的DNA复制至关重要。
值得注意的是，在复制应激条件下，携带N端磷酸化降解基序（phosphodegron）突变的FANCD2，无法在FANCD2缺陷型人成纤维细胞中重建复制叉的正常推进进程。
在FBXL12缺失的细胞中，FANCD2会被困于染色质之上，进而引发复制应激、过量DNA损伤以及细胞死亡。
在人类癌症样本中，FBXL12、细胞周期蛋白E（CYCLIN E）与范可尼贫血信号通路呈正相关表达；且在高表达细胞周期蛋白E的乳腺肿瘤患者中，FBXL12的上调或扩增与患者生存期缩短显著相关。
最终，敲低FBXL12会加剧致癌基因诱导的复制应激，并通过抑制WEE1激酶，使乳腺癌细胞对药物诱导的复制应激更为敏感。
综上，本研究结果表明，FBXL12是高表达细胞周期蛋白E的癌细胞的潜在治疗靶点，可为癌症靶向治疗提供全新方向。
本研究对MDA-MB-231基底样乳腺癌细胞的RNA测序（RNA-seq）数据开展了比较基因表达谱分析。实验共设置三份重复样本：分别为FBXL12野生型（转染随机对照向导RNA（gRNA）的对照细胞）以及两株FBXL12基因敲除（FBXL12-KO）克隆。