Background Protein phosphatase 2A (PP2A) represents a family of potent tumor suppressors that are often suppressed in human cancers by upregulation of proteins that inhibit subunit assembly into active enzyme complexes. Thus, restoration of PP2A has assumed increasing importance for cancer treatment. In earlier work, we found that perphenazine (PPZ) kills leukemic T cells by activating PP2A (Gutierrez et al. 2014. J Clin Invest.). PPZ also acts to inhibit dopamine D2 receptor (DRD2) in the basal ganglia, which causes movement disorders at dosages less than those needed to kill T-ALL cells, effectively precluding "repurposing" of PPZ for the therapy of T-ALL. Hypothesis We sought to identify PPZ analogues that activate PP2A and induce apoptosis in T-ALL cells but lack the ability to bind and inhibit DRD2. Such analogues would not induce the movement disorders that have limited the usefulness of PPZ as an anti-leukemic drug. Methods PP2A is a heterotrimeric phosphatase that is assembled from three classes of subunits, encoded by two possible "A" genes, 15 "B" genes and 2 "C" genes, providing 60 distinct PP2A holoenzymes that can potentially form in the cell. To identify the key subunits of PP2A required for the antitumor activity of PPZ in T-ALL cells, we knocked out each subunit in human T-ALL cell lines and treated these cells with PPZ. Biochemical reporter assays were established to identify a phenothiazine derivative, iHAP1, that efficiently reactivates PP2A but does not inhibit dopamine signaling. iHAP1 preclinical studies included in vivo efficacy and toxicity in T-ALL xenograft models and in vitro efficacy in 248 cancer cell lines from 35 distinct tumor types. Results Using this approach, we found that knockout of each of three specific subunit genes of PP2A - PPP2R1A, PPP2CA and PPP2R5E - uniquely conferred resistance to PPZ treatment in T-ALL cell lines,. An independent Immunoprecipitation followed by western blotting indicated that all three subunits form a functional PP2A heterotrimeric holoenzyme complex in response to PPZ treatment. Narla and coworkers have published extensively about a series of compounds called "small molecule PP2 activators" or SMAPs (Sangodkar et al. 2017 J Clin Invest.). Intriguingly, we showed that activities of SMAP compounds depend on a different "B subunit" - PPP2R2A - and target different signal transduction pathways. Based on this finding, we sought to identify analogues of PPZ that more potently activate PP2A through this mechanism and kill T-ALL cells but lack inhibitory activity against DRD2. Testing more than 80 analogues of PPZ revealed a highly potent PP2A activator, iHAP1 (improvedHeterocyclic Activators of PP2A 1). iHAP1 is ten times more potent than PPZ in its ability to activate PP2A and kill tumor cells, but does not measurably inhibit dopamine signalling. iHAP1 is highly active as an antitumor drug human T-ALL xenograft models, without causing untoward movement disorders or other toxicity in vivo. Phosphoproteomics analysis followed by detailed biochemical assays revealed that the potent antitumor activity of PPZ and iHAP1 is mediated by dephosphorylation of MYBL2, a transcription factor that is essential for expression of genes whose products mediate prometaphase, and thus for cancer cell growth and survival. SMAP compounds do not dephosphorylate the same transcription factor and rather target other phosphoproteins. Thus, the potent PP2A activator iHAP1 drives three specific PP2A subunits into an active trimeric phosphatase and this drug is highly active against T-ALL and other hematologic malignancies. iHAP1 did not exhibit measurable toxicity up to 80 mg/kg/day PO or IM for 30 days in preclinical studies of human leukemia xenografts growing in immunosuppressed mice, providing a therapeutic index for this lead compound in preclinical studies. Conclusions Our findings show that small molecules promote the assembly of unique PP2A complexes with different regulatory subunits and substrates, allowing detailed structure and function studies of PP2A family members. A goal is to identify small molecules that assemble PP2A enzymes containing each of the remaining 13 regulatory PP2A subunits, thereby targeting a diverse array of substrates crucial to the pathogenesis of cancer and other diseases. Disclosures Gray: Gatekeeper: Equity Ownership; Syros: Equity Ownership; Petra: Equity Ownership; C4: Equity Ownership; B2S: Equity Ownership; Soltego: Equity Ownership; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Janssen: Research Funding; Kinogen: Research Funding; Voronoi: Research Funding; Her2llc: Research Funding; Deerfield: Research Funding; Sanofi: Research Funding. Fischer:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Deerfield: Consultancy, Research Funding; Astellas: Research Funding.