Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

中文翻译：

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化学遗传学分析揭示了拟议的 PP2A 激活剂 iHAP1 和 DT-061 的 PP2A 独立细胞毒性

蛋白磷酸酶 2A (PP2A) 是一种丰富的磷蛋白磷酸酶，可作为肿瘤抑制因子。因此，能够激活 PP2A 的化合物是有吸引力的抗癌剂。最近报道了化合物 iHAP1 和 DT-061 可以选择性地稳定特定的 PP2A-B56 复合物以介导细胞杀伤。我们无法在生化分析和全酶组成中检测到 iHAP1 和 DT-061 对 PP2A-B56 活性的直接影响。因此，我们进行了全基因组 CRISPR-Cas9 合成致死率筛选，以揭示受这些化合物影响的生物学途径。我们发现敲除有丝分裂调节剂对 iHAP1 是合成致死的，而敲除内质网 (ER) 和高尔基体成分对 DT-061 是合成致死的。事实上，我们发现 iHAP1 直接阻断微管组装在体外和体内，因此充当微管毒物。相反，DT-061 破坏了高尔基体和与这些结构相关的 ER 和脂质合成。我们的工作提供了对 iHAP1 和 DT-061 扰动导致细胞毒性的生物学途径的洞察，并认为这些化合物不能用于剖析 PP2A-B56 生物学。