Therapeutic Potential of Small Molecule Inhibitor of Wnt Signaling in Acute Myelogenous Leukemia

Wnt signaling pathway controls various cellular and developmental processes. Deregulation of this pathway, in a wide range of human solid tumors, has been well established. However, less is known about its role in etiology of hematological cancers. Some characteristics of acute myelogenous leukemia (AML) cells such as inappropriate proliferation in the absence of normal growth signals and indefinite self-renewal similar to a stem cell are thought to be due to mutations in β-catenin, the hallmark of canonical Wnt pathway, and/or activation of Wnt pathway by fusion transcription factors. For therapeutic purposes, we have screened a peptidomimetic, β-turn library of small molecules for an inhibitor of Wnt pathway. Through massive chemical modifications the lead compound CWP231904 was selected. CWP231904 showed good inhibitory activity in the Reporter Gene Assay (RGA) on Topflash promoter in SW480 and HEK293 cells. Wnt target genes, survivin and c-myc were both down regulated upon CWP231904 treatments at the level of RNA and protein in MV4-11 and HL-60 AML cells. CWP231904 treatments of both cell lines induce cleavage of caspase-3, -8, and -9 by apoptotic signals. Propidium Iodide staining of these cells show dramatic increase in Apoptotic Index. CWP231904 treatment of MV4- 11 and HL-60 cells led to degradation of β-catenin in both cell lines. Proteasome inhibitor, MG132, prevented the CWP231904 induced degradation of β-catenin in MV4-11 cells. In order to unravel the mode of action of CWP231904, we utilized Wnt3a conditioned media (CM) or an overexpressed, Estradiol (ER) inducible Dishevelled plasmid (ER-DVL-2) to activate Wnt signaling in HEK293 cell line. Immunoblotting of HEK293 cells induced with ER and treated with CWP231904 showed a nuclear migration of the phosphorylated form of DVL-2 in treated cells. Thus, our data suggest that CWP231904 targets β-catenin degradation by relocating DVL-2 from cytoplasm where it makes puncta and inactivates GSK-3β containing destruction box complex into the nucleus. Moreover, CWP231904 treatment of HEK293 cells growing in Wnt3a CM lowered the inhibitory phosphorylation of GSK3-β on Serine9 which is induced upon Wnt activation, thereby keeping GSK3-β in its active form to phosphorylate β-catenin and signal it for degradation. CWP231904 significantly reduced tumor growth in MV4-11, HL-60 and MOLM-13 xenograft models, significantly increased the life span of the SCID mice, and decreased the number of CD45⁺ AML cells in the bone marrow after engraftment of MV4-11 cells via intravenous injection withoutdetectable toxicity. Together, these results suggest that CWP231904 has therapeutic potential for the treatment of AML disease by inhibiting Wnt signaling pathway through destabilization of β-catenin.