Abstract：
The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.
摘要：
ATR激酶保护细胞免受DNA损伤和复制应激,并且代表有希望的抗癌药物靶标。ATR抑制剂（ATRi）berzosertib和gartisertib都在临床试验中，用于单一疗法或与遗传毒性剂联合治疗晚期实体瘤。我们进行了定量的磷酸-蛋白质组筛选ATR生物标志物是高度敏感的berzosertib和gartisertib，使用优化的质谱管道。筛选确定了一系列新的ATR依赖性磷酸化事件，分为三大类：i）其磷酸化对ATRi高度敏感并且可能是下一代ATR生物标志物的靶标；ii）具有已知基因组维持作用的蛋白质，以前不知道该蛋白质受ATR调节；iii）细胞作用不清楚的新靶标。第iii类靶标代表候选DNA损伤反应蛋白，考虑到这一点，对这类蛋白质进行二次筛选，以募集到DNA损伤位点。我们发现其中一种被招募的蛋白质，SCAF1以磷酸依赖性方式与RNAPII相互作用，募集需要PARP活性和与RNAPII相互作用。我们还显示SCAF1缺乏部分挽救了缺乏BRCA1肿瘤抑制因子的细胞中的RAD51负载。总之,这些数据揭示了潜在的新的ATR生物标志物和新的基因组维持因子。
