Abstract 2631: Target validation of the LMP2 inhibitor, UK-101
Authors: Wehenkel Marie , Kim Kyung Bo 
Published: February 2011

Abstract
Introduction: The purpose of this study is to determine the mechanism(s) of action of the novel LMP2 inhibitor, UK-101. LMP2 is a major catalytic subunit of the immunoproteasome, an alternative form of the constitutive proteasome, and its upregulation has been demonstrated in a variety of disease states, including cancer. Treatment with UK-101 covalently and selectively modifies the LMP2 subunit, inhibiting its catalytic function. UK-101 has also been shown to cause apoptosis in cancer cell lines, but it is not yet clear whether this apoptotic effect is mediated by the inhibition of LMP2. Since off-target effects are major roadblocks for the development of new and effective pharmaceuticals, validating the major target of UK-101 is LMP2 would assist in the further progression of this inhibitor towards preclinical trials.
Experimental Procedures: The PC3 prostate cancer cell line was used in all studies and maintained in RPMI under standard conditions. The expression of LMP2 was increased in these cell lines using a natural inducer of the immunoproteasome (interferon-gamma). The expression of LMP2 was decreased in these cell lines using pooled siRNAs obtained from Dharmacon. Expression level changes were evaluated using standard western blotting techniques as well as via immunofluorescence, using a Nikon Ti-U microscope and NIS Element Research image analysis software. The characterized cells were then exposed to UK-101, eponemycin (UK-101 parent compound), or epoxomicin (general proteasome inhibitor) for 48 hours. The effects of each of these proteasome inhibitors on cell proliferation were measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS assay). The data were plotted in GraphPad Prism® and the calculated IC50 values are the result of at least three replicates.
Data Summary: A 24 hours pretreatment of interferon-gamma was sufficient to increase the levels of LMP2 significantly and this effect was maintained for at least 96 hours. This increase in LMP2 expression resulted in an increase in the IC50 value only for the cells treated with UK-101. Cells treated with the other proteasome inhibitors showed no significant change in IC50 values. The pooled siRNA was able to significantly decrease the levels of LMP2 following a 24 hour treatment. The knockdown was most effective after a 96 hour incubation and it was maintained up to 168 hours later. Decreasing LMP2 expression via knockdown sensitized the cells to UK-101 treatment, while a greater than 95% knockdown desensitized the cells to UK-101 treatment.
Conclusions: The current data support the hypothesis that UK-101 is exerting its effect on cell proliferation mainly through the covalent modification of the LMP2 subunit of the immunoproteasome. Further studies are needed to more clearly delineate this causal relationship. If it is confirmed, this will provide the impetus to begin preclinical evaluations of this molecule in prostate cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2631.