Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061

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.