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

Abstract

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.

Small-Molecule Inhibitors Targeting the Canonical WNT Signaling Pathway for the Treatment of Cancer

“…Compound 88 was found to reduce the transcription levels of WNT target genes survivin and c-myc and result in the degradation of β-catenin in MV4-11 and HL-60 cells. 135 It was also able to decrease tumor growth in MV4-11, HL-60, and MOLM-13 xenograft mouse models, suggesting that 88 has great potential as a drug development candidate for the treatment of AML. CWP232291 (chemical structure not disclosed), 136 another analogue of 85, is also undergoing human clinical trials and to date has displayed evidence for safe and single-agent efficacy in AML patients.…”

“…As a prodrug, compound 87 is bioconverted into CWP231904 ( 88 ) via plasma alkaline phosphatase. Compound 88 was found to reduce the transcription levels of WNT target genes survivin and c-myc and result in the degradation of β-catenin in MV4-11 and HL-60 cells . It was also able to decrease tumor growth in MV4-11, HL-60, and MOLM-13 xenograft mouse models, suggesting that 88 has great potential as a drug development candidate for the treatment of AML.…”

Medicinal Chemistry Strategies for the Development of Inhibitors Disrupting β-Catenin’s Interactions with Its Nuclear Partners

“…Prodrug 63 was transformed into the active parent drug 64 (CWP231904) by plasma alkaline phosphatase . Compound 64 suppressed the expression of Wnt target genes survivin and c-myc and remarkably reduced tumor growth in MV4–11, HL-60, and MOLM-13 xenograft mouse models …”

Carboxylesterase Notum Is a Druggable Target to Modulate Wnt Signaling

“…CWP-291 ( 8 , formerly CWP232291) is a peptidomimetic small molecule drug precursor to the active metabolite CWP232204 (structures undisclosed) developed by JW Pharmaceuticals that reduces β-catenin levels in a TOP-flash reporter assay in HEK293 cells (IC 50 273 nM) and is currently in phase 2 clinical trials for acute myeloid leukemia . The active form CWP-291 binds to Sam68 (an RNA-binding protein that regulates alternative splicing of the TCF-1 transcription factor in a complex with CBP) and induces β-catenin degradation …”

“…45 48 The active form CWP-291 binds to Sam68 (an RNA-binding protein that regulates alternative splicing of the TCF-1 transcription factor in a complex with CBP) and induces β-catenin degradation. 49 Targeting Wnt Signaling in the Cytoplasm. The posttranslational acylation of Wnt ligands is considered to be a key step prior to its secretion and activation.…”