Pre-Clinical Efficacy of Combined Therapy with Novel β-Catenin Antagonist BC2059 and Histone Deacetylase Inhibitor Against Cultured and Primary AML Blast Progenitor Cells

Abstract 287

The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem and early blast progenitor cells (BPCs). Deregulated WNT signaling in AML inhibits polyubiquitylation and proteasomal degradation of β-catenin by the multi-protein complex. The inactivation of the degradation complex for β-catenin results in the preservation, nuclear translocation and activity of β-catenin in AML BPCs. This is especially notable in AML BPCs expressing FLT3-ITD, where increased activity of PI3K/AKT phosphorylates and inactivates GSK3β, thereby inhibiting phospho-degradation, increasing stability and nuclear accumulation of β-catenin. Activated FLT3 kinase has also been shown to directly phosphorylate β-catenin and promote its stabilization and nuclear localization. As a co-activator, in the nucleus β-catenin interacts with the bipartite T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which increases the expression of pro-growth and pro-survival genes, including cyclin D1, Myc and survivin. Aberrant expression of LEF1 in hematopoietic stem cells has been shown to induce AML. β-catenin is also required for the HOXA9 and MEIS1 or MLL-AF9-mediated transformation of hematopoietic stem/progenitor cells. Thus, reactivation of β-catenin is required for maintenance of leukemic transformation making it an attractive drug target in AML. Here, we determined the in vitro and in vivo anti-AML activity of BC2059 (β-Cat Pharmaceuticals), a potent, small molecule, anthraquinone oxime-analog, against cultured (HL-60, OCI-AML3 and MV4-11 cells) and primary AML BPCs with or without the expression of FT3-ITD. First, as compared to the normal human CD34+ cells, FLT3-ITD expressing AML BPCs showed increased levels of β-catenin, mostly in the nucleus, as determined by confocal immunofluorescence microscopy. Exposure to 100 nM of BC2059 (BC) induced β-catenin degradation through the proteasome, as well as attenuated the nuclear and cytoplasmic levels of β-catenin in the cultured and primary AML BPC. Chromatin immunoprecipitation with anti-β-catenin antibody demonstrated that treatment with BC2059 (BC) reduced the binding of β-catenin to the WNT response elements (WRE) in the promoter DNA of its target genes, including Myc, cyclin D1 and survivin. Estimation of the intracellular luciferase levels in AML cells transfected with the TOP/FLASH versus FOP/FLASH construct showed that treatment with BC2059 significantly reduced only the TOP-FLASH luciferase activity, indicating that BC inhibits the expression of genes with promoters containing WRE elements. This was associated with reduced mRNA and protein levels of cyclin D1, MYC and survivin. Treatment with BC dose-dependently induced apoptosis of cultured and primary AML BPCs (up to 70%), including apoptosis of the CD34+CD38-Lin- AML BPCs. In contrast, BC induced apoptosis in < 10% of normal CD34+ progenitor cells. We have previously reported that treatment with the histone deacetylase inhibitor panobinostat (PS) attenuates p-FLT3, p-AKT and p-GSK3β levels in AML BPCs expressing FLT3-ITD. Consistent with this, here, we determined that co-treatment with BC and PS (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML BPCs. Against primary AML BPCs expressing FLT3-ITD, co-treatment with the FLT3 kinase inhibitor AC220 (100 nM) further augmented BC mediated depletion of the cytoplasmic and nuclear levels of β-catenin and significantly enhanced BC-induced apoptosis (p < 0.01). This was associated with induction of BIM and p27 with depletion of MCL-1 levels. Following tail vein infusion and establishment of AML by OCI-AML3 cells in NOD-SCID mice, treatment with BC (5 or 10 mg/kg b.i.w, IV) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of the NOD/SCID/IL2Rγ-depleted mice with established human AML following tail-vein injection of primary AML BPCs expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. These findings support a compelling rationale for further development and in vivo testing of the BC-based combination with PS and FLT3 antagonist against human AML BPCs.