Disruption of CTNNB1/LEF-1 Complex by Small Molecule Inhibitors Induces Apoptosis in B-CLL Cells in Vitro and in Vivo

Deregulated WNT signaling has been implicated in variety of tumors. Previous findings suggest critical components of WNT signaling pathway (WNTs, FZD and LEF-1) are overepxressed in B-cell chronic lymphocytic leukemia (CLL) at the m-RNA level. β-catenin (CTNNB1)/LEF-1 transcription activation complex is the key component of WNT signaling which governs the expression of WNT target genes. Critical genes like C-MYC, survivin (SURV), cyclin D1 (CCND1) and LEF-1 which are also overexpressed in CLL are downstream targets of this pathway. In the current study we attempted to inhibit CTNNB1/LEF-1 interaction in CLL cells using the novel small molecule inhibitors CGP049090 and PKF115-584 and observed the survival potential both in vivo and in vitro. Substantial overexpression of LEF-1 and dephospho-CTNNB1, in CLL cells was observed when compared to healthy CD19 positive B cells and PBMCs suggests activation of WNT signaling in CLL. The LC50 calculated from 30 CLL patients was 1.18 μM for CGP049090 and 1.10 μM for PKF115-584 by the ATP based assay. This was confirmed by the DiSC assay LC50 of 0.33 μM (CGP049090) and 0.23 μM (PKF115-584) for primary B-CLL cells (n=3). Both substances showed little effects on normal PBMCs (n=5) as seen from the LC50 values of 90.92 μM and 39.88 μM for CGP049090 and PKF115-584, respectively. Time course experiments indicate activation of caspase 8, 9, 3 & 7, PARP cleavage and paralleled by downregulation of known inhibitors of apoptosis (IAPs) MCL-1, XIAP and BCL-2 leading to apoptosis. Co-immune precipitation of CTNNB1/LEF-1 complex was inhibited in the presence of CGP049090 and PKF115-584 suggesting the specificity of the interaction. Cellular uptake of PKF115-584 and cytoplasmic co-localisation with lef-1 was observed by fluorescence microscopy. In vivo studies reveal that both substances are highly tolerable and effective in eradicating tumor growth in xenografts. The tumor inhibitory rate (IRmax) was found to be 78% and 63.8% for CGP049090 and PKF115-584, respectively. These results indicate that LEF-1 is a promising target for B-CLL therapy as CGP049090 and PKF115-584 effectively induce apoptosis within micromolar concentrations in CLL cells but do not affect healthy cells at these doses. Downregulation of WNT target genes and IAPs hasten the apoptotic mechanism. Moreover, and these inhibitors are highly effective and tolerable in vivo which identifies both compounds as promising drugs for targeted therapy in CLL.