目的:PARP抑制剂(PARPi)对同源重组修复(HRR)缺陷(HRD)癌症有效。为了(重新)使HRR熟练(HRP)肿瘤对PARPi的敏感性,正在探索与其他药物的组合。我们的目的是确定细胞周期检查点激酶ATR抑制剂对PARPi敏感的机制。CHK1和WEE1。
方法:使用一组HRD和HRP细胞(包括匹配的BRCA1或2突变体和校正对)和卵巢癌腹水细胞。Rucaparib(PARPi)诱导的复制应激(RS)和HRR(γH2AX和RAD51病灶的免疫荧光显微镜,分别),细胞周期变化(流式细胞术),激活ATR,确定CHK1和WEE1(分别用于pCHK1S345,pCHK1S296和pCDK1Y15的蛋白质印迹)和细胞毒性(集落形成测定),随后调查了ATR抑制剂(VE-821,1µM)对所有这些参数的影响,CHK1(PF-477736,50nM)和WEE1(MK-1775,100nM)。
结果:Rucaparib诱导RS(3至10倍),S相积累(2倍)和ATR,CHK1和WEE1激活(高达3倍),VE-821,PF-477736和MK-1775抑制了它们的靶标,并消除了这些rucaparib诱导的HRP和HRD细胞的细胞周期变化。Rucaparib仅在HRP细胞中激活HRR,并且对HRD细胞的细胞毒性更大(60-1,000x)。VE-821,PF-477736和MK-1775阻断HRR并使HRP敏感,但对HRD细胞和原发性卵巢腹水不敏感。
结论:我们的数据表明,而不是通过废除细胞周期检查点来发挥作用,ATR,CHK1和WEE1抑制剂引起HRD表型,因此“诱导合成致死性”与PARPi。
OBJECTIVE: PARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1.
METHODS: A panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM).
RESULTS: Rucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib.
CONCLUSIONS: Our data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence \"induced synthetic lethality\" with PARPi.