BC2059: An Orally Bioavailable β-Catenin Inhibitor Potently Induces Apoptosis As A Single Agent and In Combination With Bortezomib In Multiple Myeloma

Multiple myeloma (MM) remains an incurable malignancy of plasma cells harboured in the bone marrow. Despite the introduction of new therapeutic modalities such as lenalidomide, bortezomib and autologous stem cell transplantation, for the majority of myeloma patients treatment failure is inevitable because of the emergence of drug resistance. The canonical Wnt/β-catenin signalling pathway which plays a central role in modulating the delicate balance between stemness and differentiation in several adult stem cell niches, including the haematopoietic one in the bone marrow, has been found to be dysregulated in MM. Furthermore, it has been shown that Wnt signalling activation is associated with advanced stage MM, providing a rationale to evaluate a new orally bioavailable β-catenin inhibitor BC2059 (BetaCat Pharmaceuticals, Gaithersburg,USA) in mono- and combination therapy with proteasome inhibitors.

We have shown that BC2059 induced apoptosis in 10/10 genetically heterogeneous human myeloma cell lines (HMCL) treated with dosages ranging from 50-500nM. At 72 hours the pro-apoptotic effect of BC2059 at 500nM was almost uniform with cell death (>80%) for all the HMCLs tested. BC2059 was able to inhibit the proliferation of all HMCLs in a dose and time dependent manner assessed by MTS assay and viable cell enumeration with trypan blue. Further analyses demonstrated the accumulation of cells in the G₀/G₁ phase suggesting G₁ cell cycle arrest. The expression of the active nuclear β-catenin was confirmed by immunoblotting the nuclear fraction of the HMCLs. Furthermore we were able to demonstrate reduction of β-catenin in both cytoplasmic and nuclear compartments after 14 hours of treatment with increasing concentrations of BC2059 (for example KMS-18 treated with IC₅₀, 2xIC₅₀ and 4xIC₅₀ showed a relative drop of nuclear β-catenin from 1 in the untreated cells to 0.6, 0.5 and 0.4 respectively) suggesting that the drug facilitates its degradation. MM is well known to be dependent on the bone marrow microenvironment for its survival and drug resistance. To test the efficacy of BC2059 in overcoming this protective effect we treated HMCLs cocultured with an immortalised human stromal cell line (HS5) and we were able to demonstrate that BC2059 abolished the stromal effect at dosages > IC₈₀ (for KMS-11 with IC₅₀=215nM at 100nM the difference in cell death between myeloma cells alone and in co-culture is statistically significant, p<0.01, whereas at 350nM the difference is not significant, p>0.05). It has been already shown that bortezomib augments the activation of canonical Wnt signaling by preventing β-catenin protein from proteosome-mediated degradation in MM cells. Nevertheless, it is not clear if this effect is pro-apoptotic or pro-survival providing us a rationale to use BC2059 with bortezomib in combination therapy. 5 HMCLs were tested with concomitant administration of BC2059 and bortezomib with 4/5 showing synergistic effect (Combination Index[CI] 0.4-1.1, CI< 1.1 is considered synergistic). As a monotherapy, BC2059 effectively killed primary MM tumour cells from relapsed and/or refractory MM patients (n=13) in an autologous bone marrow (BM) co-culture assay (46±7.5% and 59±5.6% killing at at 1µM and 5µM, respectively) and demonstrated a synergistic effect when combined with bortezomib in 3 primary MM samples (Synergy Quotient[SQ]: 2.3-8.3, SQ>1 is considered synergistic). Interestingly one sample derived from a bortezomib-resistant relapsed MM patient could be re-sensitised to bortezomib by BC2059 suggesting that up-regulation of β-catenin may be one of the mechanisms of acquired drug resistance to bortezomib. In conclusion, BC2059 induces apoptosis in HMCL and primary MM cells at therapeutically relevant concentrations and is capable of overcoming the protective influence conferred by the bone marrow microenvironment.