Colis Laureen C, Hegan Denise C, Kaneko Miho, Glazer Peter M, Herzon Seth B
†Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.
‡Departments of Therapeutic Radiology and Genetics, Yale School of Medicine, New Haven, Connecticut 06520, United States.
J Am Chem Soc. 2015 May 6;137(17):5741-7. doi: 10.1021/ja513117p. Epub 2015 Apr 22.
(-)-Lomaiviticin A (1) and the monomeric lomaiviticin aglycon [aka: (-)-MK7-206, (3)] are cytotoxic agents that induce double-strand breaks (DSBs) in DNA. Here we elucidate the cellular responses to these agents and identify synthetic lethal interactions with specific DNA repair factors. Toward this end, we first characterized the kinetics of DNA damage by 1 and 3 in human chronic myelogenous leukemia (K562) cells. DSBs are rapidly induced by 3, reaching a maximum at 15 min post addition and are resolved within 4 h. By comparison, DSB production by 1 requires 2-4 h to achieve maximal values and >8 h to achieve resolution. As evidenced by an alkaline comet unwinding assay, 3 induces extensive DNA damage, suggesting that the observed DSBs arise from closely spaced single-strand breaks (SSBs). Both 1 and 3 induce ataxia telangiectasia mutated- (ATM-) and DNA-dependent protein kinase- (DNA-PK-) dependent production of phospho-SER139-histone H2AX (γH2AX) and generation of p53 binding protein 1 (53BP1) foci in K562 cells within 1 h of exposure, which is indicative of activation of nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. Both compounds also lead to ataxia telangiectasia and Rad3-related- (ATR-) dependent production of γH2AX at later time points (6 h post addition), which is indicative of replication stress. 3 is also shown to induce apoptosis. In accord with these data, 1 and 3 were found to be synthetic lethal with certain mutations in DNA DSB repair. 1 potently inhibits the growth of breast cancer type 2, early onset- (BRCA2-) deficient V79 Chinese hamster lung fibroblast cell line derivative (VC8), and phosphatase and tensin homologue deleted on chromosome ten- (PTEN-) deficient human glioblastoma (U251) cell lines, with LC50 values of 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively, and selectivities of >11.6 versus the isogenic cell lines transfected with and expressing functional BRCA2 and PTEN genes. 3 inhibits the growth of the same cell lines with LC50 values of 6.0 ± 0.5 and 11 ± 4 nM and selectivities of 84 and 5.1, for the BRCA2 and PTEN mutants, respectively. These data argue for the evaluation of these agents as treatments for tumors that are deficient in BRCA2 and PTEN, among other DSB repair factors.
(-)-洛马维亭A（1）和单体洛马维亭苷元[又名：(-)-MK7-206，（3）]是能诱导DNA双链断裂（DSB）的细胞毒性剂。在此，我们阐明了细胞对这些药剂的反应，并确定了与特定DNA修复因子的合成致死相互作用。为此，我们首先对人慢性粒细胞白血病（K562）细胞中1和3引起的DNA损伤动力学进行了表征。3能迅速诱导DSB，在添加后15分钟达到最大值，并在4小时内得到修复。相比之下，1引起的DSB产生需要2 - 4小时才能达到最大值，超过8小时才能得到修复。碱性彗星解旋试验表明，3会引起广泛的DNA损伤，这表明观察到的DSB是由紧密间隔的单链断裂（SSB）产生的。1和3在暴露1小时内均能诱导共济失调毛细血管扩张突变（ATM）和DNA依赖性蛋白激酶（DNA-PK）依赖性的磷酸化丝氨酸139组蛋白H2AX（γH2AX）产生以及在K562细胞中产生p53结合蛋白1（53BP1）焦点，这表明非同源末端连接（NHEJ）和同源重组（HR）修复被激活。这两种化合物在稍后的时间点（添加后6小时）也会导致共济失调毛细血管扩张和Rad3相关（ATR）依赖性的γH2AX产生，这表明存在复制应激。3也被证明能诱导细胞凋亡。与这些数据一致，发现1和3与DNA DSB修复中的某些突变具有合成致死性。1能有效抑制2型早发性乳腺癌（BRCA2）缺陷的V79中国仓鼠肺成纤维细胞系衍生物（VC8）以及10号染色体上缺失磷酸酶和张力蛋白同源物（PTEN）的人胶质母细胞瘤（U251）细胞系的生长，LC50值分别为1.5±0.5和2.0±0.6 pM，与转染并表达功能性BRCA2和PTEN基因的同基因细胞系相比，选择性分别大于11.6。3抑制相同细胞系的生长，BRCA2和PTEN突变体的LC50值分别为6.0±0.5和11±4 nM，选择性分别为84和5.1。这些数据支持将这些药剂评估为治疗BRCA2和PTEN以及其他DSB修复因子缺陷的肿瘤的药物。
