Targeted Inhibition of mTORC1 and mTORC2 by the Active-Site mTOR Inhibitors, PP-242 and OSI-027, Has Cytotoxic Effects In T-Cell Acute Lymphoblastic Leukemia

Abstract 3242

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia characterized by uncontrolled proliferation of lymphoblasts committed to the T-cell lineage. Despite improvements in therapy, 25% of children and 50%–70% of adults with T-ALL relapse and develop a polychemotherapy-resistant disorder, which carries a poor prognosis. Thus, major efforts are being made to develop targeted molecules against deregulated signaling pathways that sustain T-ALL cell growth and survival. The mTOR serine/threonine kinase belongs to two separate multi-protein complexes, referred to as mTORC1 and mTORC2. The mTORC1 complex, which is inhibited by rapamycin/rapalogs, controls mRNA translation through the phosphorylation of the translation repressor 4E-BP1, while mTORC2 could be involved in regulating proliferation and survival through phosphorylation of Akt on Ser 473. mTORC2 is usually considered to be rapamycin-resistant. Both mTORC1 and mTORC2 are activated in most of T-ALL patients underscoring these complexes as major targets for T-ALL treatment. However, the allosteric inhibition of mTORC1 by rapamycin/rapalogs has only modest effects in T-ALL cells. This could be due, among other things, to the fact that rapamycin/rapalogs are mainly cytostatic and do not dephosphorylate 4E-BP1 in preclinical models of T-ALL. Recently, however, ATP-competitive inhibitors specific for the mTOR kinase active site have been synthesized. These compounds target both mTORC1 and mTORC2 and are more active than rapamycin/rapalogs against p-4E-BP1. Here, we have explored the therapeutic potential of two active-site mTOR inhibitors, PP-242 and OSI-027, against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. OSI-027 is being now tested in clinical trials in patients with solid malignancies. PP-242 and OSI-027 affected T-ALL cell line viability as documented by MTT assays. IC₅₀ for PP-242 ranged from 0.15 to 0.50 μM at 24 hours, whereas OSI-027 was less potent (IC₅₀ ranging from 0.6 to 1.3 μM at 48 h). Of note, both the drugs were effective against CEM T-ALL cells overexpressing 170-kDa P-glycoprotein, one of the major determinants of drug-resistance. Overall, PP-242 was slightly more powerful than the dual PI3K/mTOR inhibitor, PI-103, whose IC₅₀ ranged from 0.2 to 1.0 μM. When T-ALL patient samples were examined, PP-242 displayed an IC₅₀ of 0.5–1.0 μM, while the IC₅₀ for OSI-027 ranged between 2.3 and 4.8 μM. Both the inhibitors affected to a much lower extent the proliferation of peripheral blood T-lymphocytes from healthy donors stimulated with phytohemagglutinin and interleukin-2. Remarkably, PP-242 targeted the side population of T-ALL cell lines, which might correspond to leukemic stem cells. The drugs induced both cell cycle arrest in G0/G1 phase and apoptosis, as documented by flow cytometric analysis, western blot analysis for cleaved caspase-3, and transmission electron microscopy. Western blot analysis demonstrated that the inhibitors induced a dose- and time-dependent dephosphorylation of Akt on Ser 473 (indicative of mTORC2 inhibition) as well as a dephosphorylation of mTORC1 downstream targets including Thr 389 p70S6 kinase, Ser 235/236 S6 ribosomal protein, Thr 37/46 4E-BP1, and Ser 209 eIF4E. Also a dephosphorylation of the Akt downstream target GSK3β on Ser 21/9 was observed. In contrast, rapamycin failed to dephosphorylate Thr 37/46 4E-BP1 and Ser 209 eIF4E. At variance with rapamycin, we also found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors, as attested by a reduction in the incorporation of 3H-leucine and a shift from large to small polysomes.In MOLT-4 and Jurkat cells, the inhibitors, when used at subcytotoxic concentrations, strongly synergized with vincristine (combination index = 0.2–0.6), a traditional chemotherapeutic drug currently used for treating T-ALL patients, and with the Bcl-2 inhibitor, ABT-263 (combination index =0.15-0.19). In conclusion, both the inhibitors displayed a remarkable anti-leukemic activity through inhibition of mTORC1 and mTORC2 activity. The lower cytotoxicity against healthy T-lymphocytes ex-vivo suggests a favourable therapeutic index, which emphasizes the development of active-site mTOR inhibitors as clinical candidates for therapy in T-ALL.