Abstract B249: Small molecule inhibitors of the Pim protein kinases block the growth of T-acute lymphoblastic leukemias

Abstract

Abstract The Pim protein kinases, first identified as a proviral integration site in c-Myc overexpressor mice, are increased in multiple human hematopoietic neoplasms including myeloid leukemia, diffuse large B-cell lymphoma, and T-cell lymphoma. We have developed novel benzylidene-thiazolidine-2, 4-diones (J. Med. Chem. (2009) 52:74) inhibitors of Pim kinases that kill 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. The most potent members of this chemotype have IC50s of 13 nM for Pim-1 and 2.3 M for Pim-2 while some compounds in this chemotype demonstrated selectivity's of more than 2500-fold and 400-fold for Pim-1 or Pim-2 respectively while other congeners had equivalent potency towards both isozymes. Of the 47 additional protein kinases tested, only DYRK1a was sensitive to these agents. Incubation of pre T-LBL cells with one of these Pim inhibitors, SMI-4a, induced G1 phase cell cycle arrest secondary to a dose dependent induction of p27Kip1 and translocation of this protein to the nucleus. Additionally, SMI-4a induced apoptosis in these leukemic cells through the mitochondrial pathway, and inhibited mTORC1 pathway based on decreases in phosphorylation of p70 S6K and 4E-BP1, two substrates of this enzyme. Using immuno-deficient animals, we demonstrate that treatment 5/7 days 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). These SMI-4a treated mice had no change in 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. Because we find in animals and cell culture that single agent SMI-4a treatment stimulates 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. 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. 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. Our results demonstrate that unique combinations of a potent Pim inhibitor, SMI-4a, and small molecule blockade of either the mTORC1 or ERK pathways have promise as a novel combination strategy for the treatment of human leukemia. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B249.