Targeting ß-Catenin Signaling in DLBCL with ICG-001 and PAM-1 Suppresses Cell Proliferation and Induces Apoptosis

Introduction: DLBCL is a fast-growing, aggressive form of NHL and ~30% of the patients are not cured with the current treatment regimens. Recently Wnt/ ß-catenin signaling has been identified as a new target pathway for lymphoma. To specifically target B-catenin we picked two drugs that act downstream of the Wnt/B-catenin pathway. ICG-001 inhibits TCF/β-catenin mediated transcription by competing specifically with β-catenin for CREB binding protein (CBP), but not for p300. PAM-1 is a p21-activated kinase 4 (PAK4) allosteric modulator acting as a nucleo-cytoplasmic shuttling protein. PAK4 interacts with and phosphorylates β-catenin on Ser675, promoting TCF/B-catenin transcriptional activity and stabilizing β-catenin through inhibition of its degradation. We conducted a pre-clinical study with ICG-001 and PAM-1 ß-catenin inhibitors on 16 ABC and GCB DLBCL cell lines and provided a molecular rationale for Wnt/ß-catenin directed therapy.

Material & Methods: RNAseq data from DLBCL cell lines, patient and normal B-cell samples were used to profile transcript abundance of the Wnt pathway in DLBCL. Western blotting was used to measure ß-catenin expression in 16 ABC and GCB DLBCL cell lines. Pharmacological inhibition of ß-catenin-signaling was tested using ICG-001 and PAM-1 compounds. A metabolically active cellular assay (CellTiter Glo) and flow cytometry with annexin V staining were used to determine cell viability and apoptosis. To address the specificity of the drug response, DLBCL cells were transduced with a ß-catenin/TCF pathway reporter, knocked down for ß-catenin, and canonical Wnt target expression was assessed using quantitative real-time (qRT-PCR) and Western blot.

Results: ß-catenin is expressed on all DLBCL cell lines at different levels that do not correlate with the ABC/GCB classification. The ß-catenin inhibitors ICG-001 and PAM-1 significantly reduced viability in all ABC and GCB DLBCL cell lines, regardless of DLBCL subtype. The EC₅₀ after 48h of treatment ranged from 1.4 μM to 6.3 μM for ICG001 and from 80 nM to 870 nM for PAM-1. Both compounds suppressed cell proliferation, induced apoptosis, and reduced Wnt/ß-catenin transcriptional activity.

Conclusions: Our data indicate that targeting ß-catenin pathway with ICG-001 and PAM-1 could be therapeutically beneficial to DLBCL patients.