In Vitro and In Vivo Efficacy of a Novel Orally Bioavailable Beta-Catenin Inhibitor-BC2059- As Monotherapy or in Combination with Proteasome Inhibitors in Multiple Myeloma

Background: The currently available treatment options are unlikely to be curative for the majority of Multiple Myeloma (MM) patients, emphasizing a continuing role for the introduction of investigational agents that overcome drug resistance. The canonical Wnt/β-catenin signalling pathway has been found to be dysregulated in MM, and its activation is associated with advanced stage MM, providing a rationale to evaluate the novel β-catenin inhibitor BC2059 in mono- and combination therapy with proteasome inhibitors in vitro and in vivo.

Methods and Results: We evaluated the activation status of the canonical Wnt pathway in 12 genetically heterogeneous Human Myeloma Cell Lines (HMCL) by assessing the expression of β-catenin protein in the nuclear compartment (active form). This showed that nuclear β-catenin was present in all HMCL tested and absent in plasma cells derived from a healthy donor. Moreover, additional stimulation of the canonical pathway with rhWnt3a was shown to be pro-proliferative, in contrast, no proliferation was seen with activation of the non canonical pathway following treatment with rhWnt5a. BC2059 (50nM to 500nM) induced apoptosis of all 12 HMCL and was able to inhibit the proliferation of all HMCL tested in a dose and time dependent manner assessed by MTS assay and viable enumeration with trypan blue (IC50: 53nM to 247nM). Mimicking the bone marrow (BM) microenvironment by co-culturing HMCL with the immortalised human stromal cell line HS-5, BC2059 was able to overcome the protective effect of HS-5 (for example KMS18 at IC90=220nM had no stromal pro-survival effect). Similarly, BC2059 was able to abolish the pro-proliferative effect of rh-Wnt3a or conditioned media derived from MM patients' BM when used at doses >100nM or 50nM, respectively. BC2059 facilitated the degradation of β-catenin protein in the nuclear cellular compartment ( >50% decrease of nuclear β-catenin in KMS18 treated with 1.5xIC50 when compared with untreated cells), furthermore, using a reporter assay we showed that BC2059 inhibited TCF/LEF transcriptional activity in a dose-dependent manner and decreased the transcription of axin2, a down-stream target gene of β-catenin - 78% reduction in KMS18 cells treated with 1.5x IC50 when compared to untreated controls. BC2059-induced HMCL cell death was associated with activation of both the intrinsic and extrinsic caspase-dependent apoptotic pathways, as shown by the accumulation of the activated forms of caspases 8, 9 and 3 following BC2059 treatment. However, inhibition of the caspase-pathway by the addition of caspase inhibitors (pan-caspase inhibitor Z-VAD, and caspase-3 inhibitor Z-DEVD) could not abolish the pro-necrotic effect of BC2059 or BC2059 plus bortezomib, suggesting a possible role for autophagy-induced cell death. As β-catenin undergoes proteasome-mediated destruction and has been found to increase following bortezomib treatment, we evaluated the effect of combining BC2059 with Bortezomib. The combination was synergistic for 6/8 HMCL tested (e.g. for LP1 CI:0.64-0.55, where CI<1.1=synergism). We also evaluated the effect of the combination of BC2059 with next generation proteasome inhibitors (carfilzomib and marizomib) where it was shown to have synergistic and/or additive effects (e.g. for carfilzomib LP1 CI:0.33-0.99). Single agent BC2059 effectively killed primary MM tumour cells from relapsed/refractory MM patients (n=13) and the combination with bortezomib was synergistic (n=2) with no effect on healthy peripheral blood mononuclear cells (n=4). Finally, BC2059 (10mg/kg) prolonged survival of xenografted NSG mice compared to untreated controls with no major side effects in Wnt/β-catenin dependent tissues (GI track and haematopoiesis).

Conclusion: We have demonstrated that BC2059 at nano-molar concentrations has a strong anti-MM effect both in vitro and in vivo and synergises with proteasome inhibitors. These data strongly support the clinical evaluation of BC2059 for the treatment of MM.