Leflunomide Synergizes with Gemcitabine in Growth Inhibition of PC Cells and Impairs c-Myc Signaling through PIM Kinase Targeting

Abstract

The immunosuppressive agent leflunomide has been used in the treatment of over 300,000 patients with rheumatoid arthritis. Its active metabolite, teriflunomide (Ter), directly inhibits dihydroorotate dehydrogenase (DHODH), an enzyme involved in nucleoside synthesis. We report that Ter not only shows in vitro anti-proliferative activity in pancreatic cancer (PC) cells as a single agent but also synergizes with the chemotherapeutic gemcitabine (Gem) in growth inhibition of PC cells. The growth-inhibitory effects of Ter are not solely caused by inhibition of DHODH. Through a kinase screening approach, we identified the PIM-3 serine-threonine kinase as a novel direct target. Subsequent dose-response kinase assays showed that Ter directly inhibited all three PIM family members, with the highest activities against PIM-3 and -1. The PIM-3 kinase was the PIM family member most often associated with PC oncogenesis and was also the kinase inhibited the most by Ter among more than 600 kinases investigated. Ter in PC cells induced changes in phosphorylation and expression of PIM downstream targets, consistent with the effects achieved by overexpression or downregulation of PIM-3. Finally, pharmacological inhibition of PIM proteins not only diminished PC cell proliferation, but also small-molecule pan-PIM and PIM-3 inhibitors synergized with Gem in growth inhibition of PC cells.

Figure 1

Teriflunomide Inhibits Proliferation and Induces G0 and/or G1 Cell Cycle Arrest in PC Cells Cells from the PC cell lines PANC-1, AsPC-1, MIA PaCA-2, and BxPC-3 cells were treated with 50–300 μM Ter for 24 to 72 h, as indicated. Cell growth was measured by the MTS assay. Cell cycle status was measured by propidium iodide staining followed by flow cytometry and western blotting. (A) Cells were treated with increasing concentrations of Ter for 72 h, and cell growth was measured, using the MTS assay. Results from one representative experiment are presented as means ± SD, with quadruplicate determinations. (B) Top panel: flow cytometry results for cell cycle distribution at t = 24 h for 25–200 μM Ter are presented as bar graphs; bottom panel: flow cytometry results for cell cycle distribution at t = 24 h and t = 48 h for 100 and 200 μM Ter are shown as histograms. (C) Cells were treated for 48 h at increasing Ter concentrations (50–200 μM) prior to cell lysis and immunoblotting with the indicated cell-cycle-related antibodies.

Figure 2

Teriflunomide Synergizes with Gemcitabine in Growth Inhibition of PC Cells The growth-inhibitory effect of teriflunomide in PC cells cannot be fully explained by inhibition of DHODH. (A) For two-drug combination experiments, PANC-1, MIA PaCA-2, AsPC-1, and BxPC-3 cells were treated with Ter and Gem for 72 h, as single agents and in combination, at constant ratios, on the basis of the previously calculated IC₅₀ values for each drug. Quantitative analysis of dose-effect relationships was determined after measurement of cell growth using the MTS assay. Potential synergistic or additive effects were calculated using CompuSyn software (Cambridge, UK). Isobolograms and combination index (CI) plots (not shown) were created, and CI values were calculated. Drug synergism, addition, and antagonism effects were defined by CI values of <1.0, 1.0, and >1.0, respectively. CI values for effective doses 50 (ED₅₀), 75 (ED₇₅), 90 (ED₉₀), and 95 (ED₉₅) are shown. Results from one representative experiment are presented as means ± SD, with triplicate determinations. (B) Expression of DHODH in untreated PC cell lines (western blot, left). Effect of uridine on Ter-mediated growth inhibition of PC cell lines (bar graphs, right). The PC cell lines MIA PaCA-2 and PANC-1 cells were treated for 48 h with 25, 50, 100, or 200 μM Ter in the presence or absence of 100 μM uridine prior to measurement of proliferation using an MTS assay.

Figure 3

PIM Serine-Threonine Kinases Participate in PC Cell Growth and Are Novel Direct Molecular Targets of Teriflunomide PIM-3, a member of the PIM family of serine-threonine kinases was identified as the most inhibited direct target of Ter in an in vitro kinase screening assay (not shown). (A) An in vitro Ter dose-response kinase assay showing the effects of Ter on the kinase activity of PIMs (10 μM ATP). (B) Expression of PIM proteins in untreated PC cell lines (western blotting) (C) Proposed binding mode of Ter on PIM-3 protein. The displayed binding pose of Ter at the PIM-3 ATP-binding site resulted from 190 ns molecular dynamics simulation. The kinase backbone is displayed as a yellow ribbon, with red regions denoting the most flexible residues during simulation. Teriflunomide forms two H-bonds with G105 and I107, together with a water-bridge interaction with D189. (D) The effect of the pan-PIM inhibitor PIM447 and the PIM-3-selective inhibitor M-110 on growth of PC cell lines. MIA PaCA-2 and PANC-1 cells were treated with 1 to 30 μM of PIM inhibitor for 72 h before measurement of cell growth with the MTS assay. Results from one representative experiment are presented as means ± SD, with quadruplicate determinations.

Figure 4

Teriflunomide Inhibits PIM Downstream Signaling in PC Cells (A) MIA PaCA-2 and PANC-1 cells transduced with lentiviral particles for overexpression of PIM-3 or control lentivirus particles were subjected to western blotting with the indicated antibodies. (B) MIA PaCA-2 and PANC-1 cells transfected with PIM-3 or control siRNA for 48 h were subjected to western blotting with the indicated antibodies. (C) MIA PaCA-2 and PANC-1 cells were treated with 50 to 200 μM of Ter for 48 h and subjected to western blotting with the indicated antibodies. (D) MIA PaCA-2 and PANC-1 cells overexpressing PIM-3 or control lentivirus particles were treated with or without 100 μM Ter for 48 h, followed by measurement of cell growth with an MTS assay. Results from one representative experiment are presented as means ± SD, with quadruplicate determinations.

Figure 5

Gemcitabine Synergizes with Pharmacological PIM Inhibitors in Growth Inhibition of PC Cells (A) PANC-1 and MIA PaCA-2 cells were treated with M-110 (PIM-3) and PIM447 (pan-PIM inhibitor) for 72 h as single agents and in combination with gemcitabine, at constant ratios, on the basis of the previously calculated IC₅₀ for each drug. Quantitative analysis of the dose-effect relationships was performed after measurement of cell growth by using the MTS assay. Potential synergistic or additive effects were calculated with CompuSyn software (Cambridge, UK). Isobolograms and combination index (CI) plots (not shown) were created, and CI values were calculated. Drug synergism, addition, and antagonism effects are defined by CI values of <1.0, 1.0, and >1.0, respectively. CI values for effective dose (ED) 50 (ED₅₀), 75 (ED₇₅), 90 (ED₉₀), and 95 (ED₉₅) are shown. Results from one representative experiment are presented as means ± SD, with quadruplicate determinations. (B) Summary view of CI values at EDs 50, 75, 90, and 95 for the PC cell lines MIA PaCA-2 and PANC-1, each treated with three different gemcitabine drug combinations for 72 h.