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.