Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

Abstract

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 small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes which mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we uncover the biological pathways targeted by these compounds. We find 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 endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work demonstrates that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology.

Pharmacological PP2A reactivation overcomes multikinase inhibitor tolerance across brain tumor cell models

“…DT-061). 37 Additionally, the drug interaction was validated across multiple GB cell lines (Fig. 1I, S1C).…”

TH588 and Low-Dose Nocodazole Impair Chromosome Congression by Suppressing Microtubule Turnover within the Mitotic Spindle

“…Images were acquired using a Plan-Apochromat 63×/1.4 NA oil objective with differential interference contrast mounted on an inverted Zeiss Axio Observer Z1 microscope (Marianas Imaging Workstation, 3i-Intelligent Imaging Innovations, Inc., Denver, CO, USA) equipped with an iXon Ultra 888 EM-CCD camera (Andor Technology, Belfast, UK). Astral and spindle MT intensities were quantified using ImageJ (National Institute of Health, Bethesda, MD, USA) as described before [ 26 ].…”

“…Assembly competent tubulin was purified from porcine brain as described before [24]. Turbidity-based MT polymerization assay was performed as described before [25,26]. Briefly, indicated concentrations of the drugs or DMSO (vehicle control) were mixed with free tubulin (2 mg/mL final concentration) and MT assembly was induced by the addition of 1 mM GTP and 10% glycerol in BRB80 buffer (80 mM PIPES, pH 6.8, 1 mM MgCl 2 , and 1 mM EGTA) at 37 • C. MT polymerization was monitored by measuring the change in absorbance (340 nm) using SpectraMax iD3 (Molecular Devices, San Jose, CA, USA) microplate reader in 30 s intervals over 60 min.…”

Identification of a Novel Protein Phosphatase 2A Activator, PPA24, as a Potential Therapeutic for FOLFOX-Resistant Colorectal Cancer

“…The B subunit regulates the phosphatase activity of PP2A depending on the B isoform bound to the AC core dimer, which is formed by the A and C subunits. 12 The A and C subunits have two relatively conserved isoforms (α and β), while the B subunit, on the contrary, has 15 distinct families allowing for this diverse variability of PP2A. 9 As PP2A is responsible for the regulation of many anticancerous processes in the cell, it is understood that PP2A is frequently inactivated in cancer.…”

“…14 A recent study showcased the discovery of a phenothiazine derivative that induced a 10-fold increase in the level of PP2A activation compared to that with phenothiazine, perphenazine (PPZ). 15 Moreover, this compound, known as an improved heterocyclic activator of PP2A 1 (iHAP1), showed activity at a concentration 10-fold lower than that of PPZ when studying the growth inhibition of T-cell lymphoblastic leukemia (T-ALL). 15 Similarly, an investigation of alternative CRC therapeutics led to the discovery of an orphan molecule, NSC30049 (NSC49L).…”