Potent Antineoplastic Activity of Two Inhibitors of Lymphoid Enhancer Binding Factor-1 (LEF-1) in Chronic Lymphocytic Leukemia (B-CLL).
Authors: Gandhirajan Rajesh Kumar , Gehrke Iris , Filipovich Alexandra , Paesler Julian , Razavi Regina , Erdfelder Felix , Hertweck Magdalena , Poll-Wolbeck Simon Jonas , Uhrmacher Sabrina , Schlösser Axel , Schmitt Esther Katharin , Plickert Guenter , Hallek Michael , Kreuzer Karl Anton 
Published: October 2019

Abstract
Abstract 885
Recent studies indicate factors governing aberrant activation of WNT signaling in chronic lymphoctic leukemia (CLL) cells. Thus, there is an increased secretion of WNT ligands indicating an autocrine loop leading to the extended survival of CLL cells. Lymphoid enhancer binding factor-1 (LEF-1) is a potent transcription factor regulating the expression of several WNT induced target genes. A comprehensive gene expression profiling from two independent studies revealed that LEF-1 mRNA was ∼3,000 fold overexpressed in B-CLL when compared to its healthy counterpart. Hence LEF-1 is a transcription factor expressed exclusively in CLL cells.
The objective of this present study is to demonstrate the therapeutic benefit of inhibiting LEF-1 expression in B-CLL cells using novel small molecule inhibitors CGP049090 and PKF115-584 in vivo and in vitro. JVM-3 cells and primary CLL cells were investigated by siRNA mediated knock down of LEF-1 and viability was assayed after 16h of incubation by flow cytometry. In vitro cytotoxicity and IC50 of the two compounds was enumerated using ATP based cell viability assay. Apoptotic response was investigated in time course experiments. Specificity of the small molecules was demonstrated by co-immunoprecipitation experiments for the LEF-1/βcatenin interaction in primary CLL cells. In vivo efficacy of the small molecules inhibitors were studied using a JVM-3 subcutaneous xenograft model in nu/nu mice.
The results indicate there is a high protein expression and nuclear localization of LEF-1 and β-catenin indicating active LEF-1 mediated transcription in CLL cells, whereas LEF-1 remained undetectable in healthy B cells. Preliminary experiments of LEF-1 inhibition using siRNAs resulted in increased apoptosis indicating LEF-1 to be an important player in the survival of B-CLL cells. This observation was extended using CGP049090 and PKF-115584 as they induce both dose and time dependent cytotoxicity in B-CLL, whereas healthy B cells are not significantly affected. The IC50 for CGP049090 and PKF-115584 in CLL cells were 0.7 μM and 0.9 μM, respectively. Healthy B cells were not significantly affected, as ascertained by the fact that IC50 values could not be reached due to lacking total cell death. CGP049090 and PKF-115584 induced apoptotic cell death in primary CLL cells and cell lines by cleavage of caspases 8, 9, 3 and 7 and subsequent cleavage of poly (adenosine diphospate-ribose) polymerase (PARP). Both the inhibitors also altered the expression of several anti apoptotic proteins like XIAP, mcl-1 and bcl-2. Co-immunoprecipitation experiments revealed that both the inhibitors effectively break the β-catenin/LEF-1 interaction, resulting in down regulation of expression of LEF-1 target genes such as c-myc, cyclin D1 and LEF-1. Furthermore, the inhibitors were tested in an in vivo JVM-3 subcutaneous xenograft nude mouse model resulted in >70% inhibition of tumor growth and an increase in the median survival of the treated group without leading to systemic toxicity. Immunohistochemistry analysis of the tumor sections revealed LEF-1 down regulation paralleled by inhibition of proliferation by down regulation of Proliferating Cell Nuclear Antigen (PCNA) and increase in apoptosis (induction of cleaved PARP). In summary, we show that LEF-1 is a potential therapeutic target in the treatment of CLL. Both CGP049090 and PKF115-584 show potent inhibitory effects on the survival of CLL cells in vitro and in vivo without affecting healthy B-cells, suggesting them as potential anti-cancer agents in CLL and other neoplastic malignancies with aberrant LEF-1/TCF transcriptional activity. Further investigations are warranted to determine the feasibility of these small molecules for therapeutic approaches in humans.

Disclosures:
Schlösser: Novartis: Employment. Schmitt:Novartis: Employment. Hallek:BayerScheringAG: Honoraria, Research Funding.