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. 2014 May;16(5):403-12.
doi: 10.1016/j.neo.2014.05.004. Epub 2014 Jun 18.

A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas

Jason M Foulks  1 , Kent J Carpenter  2 , Bai Luo  1 , Yong Xu  1 , Anna Senina  1 , Rebecca Nix  1 , Ashley Chan  1 , Adrianne Clifford  1 , Marcus Wilkes  1 , David Vollmer  1 , Benjamin Brenning  1 , Shannon Merx  1 , Shuping Lai  1 , Michael V McCullar  1 , Koc-Kan Ho  1 , Daniel J Albertson  3 , Lee T Call  2 , Jared J Bearss  4 , Sheryl Tripp  5 , Ting Liu  3 , Bret J Stephens  2 , Alexis Mollard  2 , Steven L Warner  2 , David J Bearss  6 , Steven B Kanner  1
Affiliations

A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas

Jason M Foulks et al. Neoplasia. 2014 May.

Abstract

The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

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Figures

Figure 1
Figure 1
Structure and analysis of TP-3654. (A) TP-3654 compound structure is shown. (B) Selectivity analysis of TP-3654. IC50 values are shown for the most potently inhibited kinases. PIM kinase values are Ki values, which were comparable to the IC50 determinations. (C) The PIM-1–specific cellular EC50 of TP-3654 was determined in a phospho-BAD (S112) Surefire (PerkinElmer, Waltham, MA) assay using HEK-293 cells transfected with BAD and PIM-1. The graph represents data from a single experiment, where the EC50 values from four independent experiments (average = 67 nM) were determined using GraphPad Prism software.
Figure 2
Figure 2
PIM-1 overexpression increases cellular pBAD levels, whereas TP-3654 decreases pBAD levels and PIM-1–driven xenografts. (A) HEK293T cells were transfected with empty vector, PIM1, and PIM1 kinase dead (PIM1KD, K67M) alone or in combination with the PIM substrate BAD using Effectene (Qiagen). Twenty-four hours posttransfection, cells were serum starved overnight and lysed for Western blot detection of phospho-BAD (pBAD) at Ser112 using a Li-Cor Odyssey scanner (Lincoln, NE). (B) UM-UC3 cells were treated for 12 hours with 3, 1, 0.3, and 0.03 μM TP-3654. Separate, identical blots were treated with antibodies to measure levels of S112 phosphorylated BAD, total BAD, Th37/46 phosphorylated 4EBP1, and total 4EBP1. (C) Parental 22RV1 and 22RV1/PIM-1 cells (5 × 106) were implanted per Nu/Nu mouse, with 10 mice per group. Mice were dosed by intraperitoneal injection with 25 mg/kg SGI-9481 or vehicle, QD × 3 weeks, 5 days on 2 days off. Tumor measurements by caliper and body weights (data not shown) were obtained. P values for caliper and body weights were *P = .023 and P = .0002, respectively. (D) Parental NIH-3 T3 and NIH-3 T3/PIM-2 cells (5 × 106) were implanted per Nu/Nu mouse, with 10 mice per group. Mice were dosed by intraperitoneal injection with 25 mg/kg SGI-9481 or vehicle, QD × 3 weeks, 5 days on 2 days off. Tumor measurements by caliper and body weights (data not shown) were obtained. P values for caliper and body weights were *P = .002 and P = .0001, respectively.
Figure 3
Figure 3
Validation of PIM-1 in solid tumor models in vitro. (A) PIM-1 shRNAs or control shRNA (nontarget) was transfected into the UM-UC-3 bladder carcinoma cell line, and PIM-1 mRNA, PIM-1 protein, and cell proliferation in a two dimensional colony formation assay were evaluated. Cells were infected with lentiviral particles overnight, the media were changed, and the cells were collected at 48 hours posttransduction for RNA or protein. A portion of cells were seeded in a six-well plate at 500 cells per well. Cells were fixed and stained after 10 days of growth. Data for PIM-1 mRNA levels are the average (±SD) of two independent experiments, whereas Western blot and colony formation assays are representative of three independent experiments. *P = .0028 and **P = .0002. (B) T24 (bladder) or (C) UM-UC3 (bladder) cancer cell lines were seeded at (B) 300 or (C) 500 cells per well in a 12-well plate and treated the next day with titrated concentrations of TP-3654 as indicated. Cells were grown for 10 or 6 days, respectively, stained for imaging, and lysed for quantitation by absorbance at 560 nm. Data are representative of independent experiments evaluating TP-3654 using T24 and UM-UC3 cell lines, with an average EC50 = 1.1 ± 0.4 μM (n = 4) and 2.2 ± 0.2 μM (n = 2), respectively. Colorimetric quantification of cell growth was performed and is provided as the graphs below the respective images with respective EC50 values.
Figure 4
Figure 4
PIM2 kinase expression in urothelial carcinoma cases. (A) Immunohistochemical stained sections of noninvasive low-grade papillary urothelial carcinoma (×200) are shown. (B) Noninvasive high-grade urothelial carcinoma (400×) is shown. (C) Invasive high-grade urothelial carcinoma (×400) is shown. (D) No expression in an invasive high-grade urothelial carcinoma (×400) is shown.
Figure 5
Figure 5
TP-3654 inhibits the growth of established solid tumor xenografts. (A) UM-UC-3 bladder carcinoma cells (5 × 106) were implanted per Nu/Nu mouse, with 12 mice per group. Mice were dosed with TP-3654 orally at 200 mg/kg QD, 3 weeks, 5 days on 2 days off or with vehicle. Caliper measurements (left panel) and tumor weights at the end of the study (middle panel) are shown. No significant change in body weights was observed (right panel). *P = .0028 and **P = .02. (B) PC-3 prostate adenocarcinoma cells (7.5 × 106) were implanted per male Nu/Nu mouse, with 12 mice per group. Mice were dosed with TP-3654 orally at 200 mg/kg QD, 3 weeks, 5 days on 2 days off or with vehicle. Caliper measurements (left panel) and tumor weights at the end of the study (middle panel) are shown. No significant change in body weights was observed (right panel). *P = .007 and **P = .0002.
Figure 6
Figure 6
Oral pharmacokinetic data for TP-3654. Plasma levels of TP-3654 (ng/ml) in female SD rats were determined using liquid chromatography-mass spectrometry. Rats were dosed either by IV injection at 2 mg/kg or orally at 40 mg/kg animal body weight. A simple formulation consisting of 10% polysorbate 20 resulted in the highest exposure (or area under the curve) and showed the highest bioavailability when compared to the IV injected animals.
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