Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia
Alejandro Gutierrez, … , A. Thomas Look, Jon C. Aster
Alejandro Gutierrez, … , A. Thomas Look, Jon C. Aster
Published January 9, 2014
Citation Information: J Clin Invest. 2014;124(2):644-655. https://doi.org/10.1172/JCI65093.
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Research Article Oncology

Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia

Abstract

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug’s antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

Authors

Alejandro Gutierrez, Li Pan, Richard W.J. Groen, Frederic Baleydier, Alex Kentsis, Jason Marineau, Ruta Grebliunaite, Elena Kozakewich, Casie Reed, Francoise Pflumio, Sandrine Poglio, Benjamin Uzan, Paul Clemons, Lynn VerPlank, Frank An, Jason Burbank, Stephanie Norton, Nicola Tolliday, Hanno Steen, Andrew P. Weng, Huipin Yuan, James E. Bradner, Constantine Mitsiades, A. Thomas Look, Jon C. Aster

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Figure 2

Results of a screen for small molecule inhibitors that synergize with the γ-secretase inhibitor compound E to suppress the growth of the human T-ALL cell line KOPT-K1.

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Results of a screen for small molecule inhibitors that synergize with th...
KOPT-K1 cells were incubated in 384-well format with small molecules from the Broad Institute bioactives collection in the presence (A) or absence (B) of GSI (compound E, 0.1 μM) in duplicate. The effects of drugs on cell growth were assessed after 2 days by incubating with CellTiter-Blue (Promega)for an additional 4 hours, and each replicate compound measurement was converted to a Z score relative to the corresponding DMSO-control distribution, as described for ChemBank (70). (C) ChemBank composite Z scores were computed to combine replicates within each condition (+GSI and –GSI), allowing the assays to be considered in a single plot; here, we sought compounds that were synergistic with GSI, i.e., they score with negative composite Z in the presence of GSI and have less effect in the absence of GSI. To express these preferences quantitatively, we devised a 2-assay score (D) that scales the +GSI composite Z both by the extent to which it points in the negative direction and by the extent to which it exceeds the –GSI composite Z (see Methods); 330 compounds tested met our initial criterion (scale factor > 0.5), of which 43 compounds, including PPZ (inset), were statistically distinguishable from the DMSO-control distribution. See Methods and Supplemental Figure 4 for additional details; screen data are available in ChemBank (see Methods).