Synergistic Killing of Leukemic Cells by Small Molecule Inhibitors of the Pim Protein Kinases and Inactivators of Additional Signal Transduction Pathways.

Abstract 409

The serine/threonine Pim kinases are up regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by c-Myc, N-Myc, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. Pim protein kinases were first identified as a proviral integration site in c-Myc overexpressor mice and function to greatly enhance lymphoma development. Here we demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases supplied by Vortex Biotechnology (Mt. Pleasant, SC), kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre T-LBL/T-ALL) being the most sensitive. Incubation of pre T-LBL cells with SMI-4a induced G1 phase cell cycle arrest secondary to a dose dependent induction of p27Kip1, apoptosis through the mitochondrial pathway, inhibition of mTORC1 pathway based on decreases in phosphorylation of p70 S6K and 4E-BP1, two substrates of this enzyme, and down regulation of c-myc. We demonstrate that treatment with 60 mg/kg twice daily by oral gavage of SMI-4a inhibits subcutaneous growth of pre T-LBL tumors by an average of 47.9% (p< .05) in immuno-deficient animals without notable toxicity to weight, blood counts, cell morphology, or blood chemistries. To enhance the killing effect of SMI-4a we have examined a number of potential combination therapies. First, because we find in animals and cell culture that single agent SMI-4a treatment up regulates the ERK pathway and in the spleen and thymus of Pim1/2/3 knock out mice there is increased phosphorylation of ERK1/2, we combined SMI-4a and a MEK1/2 inhibitor, U0126 or PD184352. Our results demonstrate that this combination is highly synergistic in killing pre T-LBL cells in culture. Secondly, because SMI-4a shares a number of important properties with γ-secretase inhibitors (GSI), Notch1 pathway inhibitor, including inhibition of pre T-LBL cell growth, cell cycle arrest, induction of p27Kip1, mTORC1 inhibition, and c-Myc down regulation, we tested the possibility that these agents could be synergistic. We find that single agent treatment with SMI-4a at 5 μM or treatment with the GSI Z-IL-CHO at 10 μM kills less than 20% of pre T-LBL cells, whereas in combination these drugs kill 78% of these cells, suggesting a high degree of synergy. Finally, because SMI-4a inhibits the mTORC1 pathway decreasing the phosphorylation of two mTOR substrates, p70 S6K and 4E-BP1, and because Pim plays an essential role in the FLT3/ITD signaling pathway, we examined the activity of SMI-4a with or without rapamycin in myeloid leukemic MV4-11 carrying both MLL-AF4 and FLT3-ITD and the RS4-11 cell line containing only MLL-AF4. We find that these two agents are highly synergistic in culture. SMI-4a alone inhibited growth 18% and rapamycin 40% but when combined 76% of the cell growth was blocked. SMI-4a had no effect on RS 4-11 cells. Our results demonstrate that unique combinations of a potent Pim inhibitor, SMI-4a, and small molecule blockade of either the mTORC1, ERK or Notch pathways has promise as a novel combination strategies for the treatment of human leukemia.