Modulation of the ATM/autophagy pathway tips the balance between senescence and apoptosis in response to 20A ligand

G-quadruplex ligands (G4L) exert their anti-proliferative effect through telomere-dependent and -independent mechanisms, but the inter-relationship between autophagy, cell growth arrest and cell death induced by these ligands remains largely unexplored. 20A is a 2,4,6-triarylpyridine derivative that binds to G4-DNA with fair to excellent selectivity. Here, we demonstrate that this compound impairs cancer cell viability through induction of senescence and apoptotic cell death in a p53-independent manner. In vivo results corroborate those obtained in in vitro, showing that 20A elicits an important tumor growth inhibition in HeLa-xenografted tumor model. The transcriptomic and proteomic analyses reveal the functional enrichment in the growth arrest, DDR and lysosomal pathways upon 20A treatment. More particularly, we find that ATM and autophagy are activated upon 20A treatment. Genetic inhibition of ATM following 20A treatment inhibits both autophagy and senescence and directs cells to apoptosis. Moreover, loss of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation and increased cell death triggered by 20A. Our results therefore identify ATM as a critical determinant in the balance between senescence and apoptosis and uncover autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal therapeutic effect of the 20A G4-ligand. Hela cells were treated or left treated with the G4 ligand 20A . At time points 6 hand 16h, total RNA were extracted for microarray experiment. Three replicates were performed at each time point of treatment and at 6h of control.

G-四链体配体（G-quadruplex ligands, G4L）通过端粒依赖与非端粒依赖的两种机制发挥抗增殖效应，但此类配体诱导的自噬（autophagy）、细胞生长阻滞与细胞死亡之间的相互关联仍未得到充分探索。20A是一种2,4,6-三芳基吡啶衍生物，对G-四链体DNA（G4-DNA）具有中等至优异的结合选择性。本研究证实，该化合物通过p53非依赖的方式诱导细胞衰老与凋亡性细胞死亡，从而削弱癌细胞的生存能力。体内实验结果与体外实验结果一致，表明20A在HeLa细胞异种移植瘤模型中可显著抑制肿瘤生长。转录组与蛋白质组分析显示，经20A处理后，样本在细胞生长阻滞、DNA损伤应答（DNA Damage Response, DDR）以及溶酶体通路中出现功能富集。更具体地说，我们发现经20A处理后，毛细血管扩张性共济失调突变激酶（Ataxia Telangiectasia Mutated, ATM）与自噬均被激活。在经20A处理后对ATM进行遗传学抑制，可同时阻断自噬与细胞衰老，并促使细胞走向凋亡。此外，通过敲除两个关键自噬基因ATG5与ATG7以丧失自噬功能，会导致检查点激酶1（CHK1）激活失败，并加剧20A诱导的细胞死亡。因此，本研究结果证实ATM是调控细胞衰老与凋亡之间平衡的关键决定因子，并揭示自噬是该调控过程的核心介质之一。因此，靶向ATM/自噬通路或许是最大化发挥20A这类G-四链体配体治疗效果的可行策略。将HeLa细胞经G-四链体配体20A处理或不予处理，分别于处理后6小时与16小时提取总RNA以开展基因芯片实验。各处理时间点均设置3次重复，6小时时间点的对照组亦设置3次重复。