Project Details

Synthesizing novel activators of PP2A in T-ALL cells

Ken Morita, MD, PhD

Summary

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most prevalent cancer among children in the U.S. and about 6,000 new cases are reported annually. T-cell ALL (T-ALL) comprises 15% of ALL cases and forms a subgroup at high risk for treatment failure. GAP: Despite continuous advances in the treatment protocols, T-ALL patients with unfavorable pretreatment signs or with recurrent disease still have a dismal prognosis and novel treatment options are needed. HYPOTHESIS: We have recently identified perphenazine (PPZ) as a FDA-approved drug that unexpectedly kills T-ALL cells through its activation of protein phosphatase 2A (PP2A), which is a critical tumor suppressor. Yet, because of its other activities as an antagonist dopamine in the brain, PPZ can cause serious movement disorders and extrapyramidal symptoms, limiting its potential as an anti-leukemic agent. In this research, I will develop orally available analogues of PPZ with increased potency against T-ALL cells and with reduced off-target neurologic side effects. METHODS: I will identify PP2A subunits responsible for PPZ’s activity in a cell-based assay. I will then clarify the effects of PPZ on PP2A activity and dopamine receptor antagonism in biochemical assays. Based on the findings, I will synthesize analogues of perphenazine and test them in vivo in zebrafish T-ALL models and also in vitro in human T-ALL cells. These experiments will be done under the supervision of Dr. Eric Fischer, Dr. Nathanael Gray and Dr. A. Thomas Look. RESULTS: For the past two years, I have worked to separate the ability of PPZ to activate PP2A and kill leukemia cells from its effects on dopamine receptors that lead do dose-limiting toxicity. By combining biochemical reporter assays that assess a panel of 91 PPZ-related compounds for their i) potency to activate PP2A, ii) potency to kill KOPT-K1 T-ALL cells, and iii) inhibitory activity to inhibit dopamine receptor signaling, I have successfully identified an analogue of PPZ, which is designated as iHAP1 (improved heterocyclic activators of PP2A), as a compound that is 10 times more potent in activating PP2A with no detectable inhibitory activities on dopamine receptor signaling. I also discovered that iHAPs potently activate a specific PP2A complex comprised of PPP2R1A (scaffold), PPP2CA (catalytic) and PPP2R5E (B56e; regulatory). iHAP1 showed excellent anti-leukemic activity both in vitro and in vivo using multiple distinct T-ALL cells lines and T-ALL xenograft preclinical mice models, respectively. Moreover, iHAP1-treated mice did not develop phenothiazine-related serious movement disorders or any other toxicity such as myelosuppression at dosages up to 80 mg/kg per day for 30 days, providing a wide therapeutic window for the use of this compound in tumor-bearing animals. IMPACT: iHAP1, the most active PPZ analogues in killing T-ALL cells that have the least CNS toxicity that has been discovered during this grant project, will be prioritized for further preclinical studies. The best molecule will then be developed as candidates for testing in clinical trials of pediatric cancer patients.

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