Disrupting Wnt/ß-Catenin Signaling By a ß-Catenin/CBP Modulator C-82 Suppresses Cell Growth and Induces Apoptosis in AML Blasts and CD34⁺38^- AML Progenitor Cells

Wnt/ß-catenin signaling has been reported to be active in AML and essential for AML stem cells but not absolutely required for self-renewal of adult hematopoietic stem cells. Targeting ß-catenin may represent a novel therapeutic opportunity for AML that should be further investigated in clinical studies.

Using reverse-phase protein array, we determined ß-catenin expression in samples obtained from 511 newly diagnosed AML patients and found that high ß-catenin level is associated with unfavorable cytogenetics (p = 0.01). Expression of ß-catenin is correlated with its target gene survivin in these samples (n= 511, R=0.24, p < 0.0001). In paired samples, ß-catenin expression is significantly higher in bone marrow (BM) than peripheral blood samples (n=140, p < 0.001) and in relapsed compared to newly diagnosed samples (n=47, p = 0.04).

C-82, like ICG-001 is a ß-catenin/cAMP-response element-binding protein (CBP) modulator which binds to CBP and inhibits the interaction between ß-catenin and CBP thus disrupts Wnt/ß-catenin/CPB mediated cell proliferation/self-renewal signaling. In the present study, we investigated the effects of inhibition of ß-catenin/CBP by C-82 in AML cells on cell growth, cell survival, the expression of the downstream targets, and the therapeutic potential of C-82 in AML.

Treatment with C-82 suppressed cell growth in AML cell lines. Cell cycle analysis showed that the treatment inhibited Edu incorporation and greatly decreased S-phase cells accompanied with increased G1 and subG1 cells in OCI-AML3 and Molm13 cells. Significant cell death was observed in C-82 treated OCI-AML3 and Molm13 cells (EC₅₀=0.82±0.04 μM and 0.79±0.02 μM, IC₅₀=0.42±0.06 μM and 0.39±0.02 μM, respectively) at 48 hours and cells from AML patients (n=7, EC₅₀=1.90±0.28 μM and IC₅₀=1.17±0.26 μM) including CD34⁺38^- AML stem/progenitor cells (n=6, EC₅₀=4.25±0.69 μM and IC₅₀=2.11± 0.04 μM) at 72 hours, while minimal cell death was found in CD34⁺ cells from normal BM (n=3, <10% with 2.5 μM C-82 at 48 hours). Decreased expression of survivin, c-Myc, and CD44, all downstream of ß-catenin/CBP signaling were found in cell lines, bulk primary AML and CD34⁺38^-cells (n=7) by western blot or flow cytometry analysis.

Co-cultures of AML cells with BM-derived mesenchymal stromal cells (MSCs) induced ß-catenin and the adhesion protein CD44, a target of ß-catenin in AML cells suggesting that the BM microenvironment regulates ß-catenin expression in AML cells. This finding is supported by the observed higher ß-catenin levels in BM compared to peripheral blood cells. C-82 suppressed MSC-induced CD44, inhibited the adhesion of OCI-AML3 cells to MSCs, and induced apoptosis of AML cell lines and blasts (n=5) and CD34⁺38^-cells (n=4) from AML patients co-cultured with MSCs. Furthermore, combination of C-82 with Ara C further enhanced death of AML cells (combination index < 1).

The data demonstrate that disrupting Wnt/ß-catenin signaling by a ß-catenin/CBP modulator C-82 suppresses cell growth and induces apoptosis in AML cells including CD34⁺38^- AML stem/progenitor cells while it has minimal toxicity in normal CD34⁺ cells supporting the clinical development of the strategy to test the therapeutic potential of targeting wnt/ß-catenin signaling in AML. PRI-724, a C-82 pro-drug is currently in clinical trial as a single agent and in combination with Ara C in patients with advanced AML.