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.

Lysosomal Changes in Mitosis.

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.