Overcoming Innate Resistance to a Beta-Catenin Inhibitor-BC2059- By Manipulating Autophagy in Multiple Myeloma

Introduction: Chemoresistance is a major challenge in the development of new therapies in multiple myeloma [MM]. Inhibition of autophagy has been shown to restore chemosensitivity in several tumors. We have previously validated a beta-catenin inhibitor (BC2059) which targets the Wnt/beta-catenin signaling pathway.

Aim: In the present study we aim to overcome innate resistance to BC2059 by manipulating the autophagic pathway.

Methods: Autophagic flux was estimated by measurement of LC3II/LC3I in the absence and presence of hydroxychloroquine [HQ] by Western blot [WB]. Induction of autophagy was measured by the increase of LC3II/LC3I by WB, and concomitant drop of p62 expression by Flow Cytometry [FC]. Combination Indices [CI] were calculated using Calcusyn software.

Results: BC2059 induces apoptosis in a dose-dependent manner by induction of both the intrinsic and extrinsic apoptotic pathways, (increase of active -caspase-8, -caspase-9 and -caspase-3 measured by FC and cleaved PARP by WB) in all human myeloma cell lines [HMCL] tested. All HMCL tested have significant autophagic flux at baseline. Chemical inhibition of autophagy has an anti-proliferative effect, decreasing the relative cell numbers from 40% (NCI-H929) to 23% (KMS12BM) at 24hr. In parallel BC2059 is able to induce autophagy in a dose dependent manner. Induction of autophagy is BC2059 specific as treatment with melphalan or bortezomib at relative equal anti-proliferative doses did not increase LC3II/LC3I. Further autophagic inhibition by HQ was synergistic for all HMCL with CI of 0.7-0.3 (CI<1.1 indicates synergy). Interestingly, inhibition of autophagy halved the LD₅₀ of BC2059 in a resistant HMCL (LP1). Other autophagy inhibitors (3-MA, bafilomycin A1 and NH₄Cl) were also synergistic with BC2059 in LP1.

Conclusion: BC2059 exerts cytotoxicity mainly by induction of apoptosis but also induces cyto-protective autophagy. Autophagy inhibition was able to overcome innate resistance to the drug, ameliorating its cytotoxic effect. This study warrants further investigation.