The Antimalarial Agent Artesunate Overcomes Bortezomib Resistance in Myeloma Cell Lines Through Non-Caspase Mediated Apoptosis
Authors: Papanikolaou Xenofon , Tytarenko Ruslana , Garg Tarun K. , Johnson Sarah K. , Tian Erming , Apewokin Senu , Barlogie Bart , Epstein Joshua , Heuck Christoph 
Published: October 2019

Abstract
Abstract 4015
During terminal differentiation of a B-lymphocyte to a mature plasma cell, the protein load increases considerably while the proteasome capacity decreases. The fact that plasma cells operate close to the limit of their proteasomal capacity is being exploited therapeutically and proteasome inhibition has become one of the pillars of myeloma treatment. The importance of the proteasome in Multiple Myeloma (MM) has also been established in prognostic terms (1). De novo or acquired resistance to proteasome inhibitors (PI) confers poor prognosis in MM. In vitro bortezomib resistance is accompanied by cross-resistance to other known PIs, thus highlighting (2), the need for treatment alternatives.
Based on reports that various antimalarial drugs exhibit in vitro anti-MM activity through either α-ring proteasome inhibition (mefloquine) or aggresome formation inhibition (quinine), we examined whether Artesunate (ART), a new highly effective antimalarial drug with a known excellent safety profile, could overcome resistance to PI's currently in clinical use.
Bortezomib (BZ)-resistant sublines of the BZ naïve and sensitive JJN3 and U266 cell lines were developed by exposure to progressively increasing BZ concentrations. Eventually the JJN3BR and U266BR sublines were developed with a 20- and 25-fold IC50 increase, respectively, compared to their BZ naïve counterparts. ART was able to inhibit viability in clinically achievable dosages and in a time-dependent manner in IL-6-independent and -dependent MM cell lines (Table 1). JJN3BR and U266BR showed no evidence of cross-resistance compared to their parental cell lines. The RPMI 8226 cell line known to exhibit one of the highest BZ IC50s among MM cell lines proved to be among the most sensitive to ART. The in-vitro antiMM effectiveness of ART was evident also in primary CD-138+ MM cells from six (6) patients with Relapsed/Refractory MM (Table 2). ART in-vitro anti MM activity was also verified by Annexin-V and 7-AAD flow cytometry for its ability to induce both early and late apoptosis.
To elucidate ART's mode of action, we measured the sequence and level of caspase activation through the luminescence assay Caspase-Glo. ART exposure initiated the extrinsinc pathway of apoptosis with a brisk increase in activated caspase-8 levels and effector caspase3/7 levels within 3h, followed by caspase 9 activation at 6h. However, actual apoptosis by ART was not accompanied by a marked caspase activity especially when compared to other potent anti-MM agents. The pan-caspase inhibitor Z-VAD-MFK effectively inhibited caspase activation by ART but failed to inhibit ART's effect on viability, as at least 70% of ART's effect was retained in JJN3, U266 and RPMI 8226 cells. Similar results were obtained in flow cytometry-based apoptosis assays. Immunoblotting of the mitochondrial derived non-caspase mediating apoptosis factors AIF, EndoG and HtrA2 in ART treated JJN3 cells revealed that ART-mediated apoptosis was related to the cytoplasmic and subsequently nuclear translocation of AIF.

Conclusion:
ART shows marked anti-MM activity and overcomes BZ resistance in vitro. Its benign side effect profile support its potential for myeloma therapy. Most anti-cancer agents induce apoptosis in a caspase-dependent manner. ART's mode of action is mainly caspase-independent, indicating that its role in overcoming drug resistance, could also extend to other anti-MM agents. Experiments aimed at further elucidating the mechanism of action of ART and the evaluation of drug combinations including ART are currently under way.

Disclosures:
Tian: University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties.