The Antimalarial Agent Artesunate Exerts Its Antimyeloma Activity By Affecting The Mitochondria and The Reactive Oxygen Status Of The Myeloma Cells and Its Efficacy Depends On Intracellular Bivalent Iron Levels
Authors: Papanikolaou Xenofon , Johnson Sarah K. , Garg Tarun K. , Tian Erming , Tytarenko Ruslana , Barlogie Bart , Epstein Joshua , Heuck Christoph 
Published: October 2019

Therapeutic advances have resulted in a 5 year life expectancy for patients diagnosed with multiple myeloma (MM) of 70% compared to 15% two decades ago. However MM remains incurable for a majority of patients and ultimately resistance to the most effective antimyeloma agents develops in the clinical setting of a late MM relapse.
The antimalarial artesunate (ART) exerts potent antimyeloma activity in vitro by inducing a non-caspase mediated apoptosis without cross-resistance to other antimyeloma agents (Blood, Nov 2012; 120:4015). Non caspase mediated apoptosis is driven by the nuclear translocation of the mitochondrial factors AIF and EndoG which occurs in myeloma cells after ART treatment, as we have previously shown. Hence, we hypothesized that mitochondria constitute a primary target of ART in MM cells. Therefore we examined the effect of ART on the mitochondria by examining the mitochondrial membrane potential (ΔΨm) of the JJN3, RPMI and INA-6 myeloma cell lines, using the JC-1 dye as an indicator of the change in ΔΨm. ART produced a small but statistically significant change in the ΔΨm as early as 30 minutes after drug exposure (ΔΨm=0.222 vs. ΔΨm=0.327 p=0.021). This change held constant for 12 hours when a brisk and continuous increase in the ΔΨm was noticed.
Mitochondria play a pivotal role in the cellular ROS equilibrium and high levels of ROS are linked to the induction of apoptosis. We therefore proceeded with examining the effects of ART on the ROS status of the cells and on mitochondrial derived superoxide production. ART at 125 μΜ (IC90) caused an initial ROS increase within 30 minutes (829 vs. 701 fluorence units (FU) mean value, p=0.043); however, the ROS level decreased with continued drug exposure, reaching subnormal levels at 12 hours and afterwards (701 vs. 433 FU mean value, p<0.001). In contrast, bortezomib (BZ) at comparable effective dosage (10nM, IC90) produced ROS levels around normal at the 12h timepoint (701 vs 750 FU mean value, p=0.176). This decrease cannot be explained by the reduced number of viable cells, since early apoptosis, as measured by annexin V expression using flow cytometry, could only be detected after 12 hours. While subnormal ROS levels were detected by the general ROS stain H2DCFDA, staining with the mitochondria specific dye MitoSOX™ revealed high levels of mitochondrial derived superoxide as measured by flow cytometry.
An iron dependent mechanism has been proposed for the antimalarial action of ART. We therefore examined the effect of iron on ART’s anti-MM efficacy. Supplementation of growth medium with 0.8 mg/L bivalent iron in the form of Iron (II) sulfate heptahydrate (a concentration resembling the normal iron concentration in human blood ) drastically increased ART efficacy as evidenced by a marked decrease in the IC50 of ART in the JJN3, INA-6, RPMI-8286, cell lines. The protein bound form of iron, holotransferrin, was not as effective in reducing the IC50, which did not achieve statistical significance when the trivalent form of iron was used (Table 1). Deferoxamine, a cell membrane permeating metal chelator with a specific affinity for iron, exerted clear antagonism to ART since IC50 could not be reached. However when the non cell membrane permeating metal chelator EDTA was used there was not a statistical significant effect ART’s IC50 in JJN3 and RPMI-8226 cells, suggesting that it is the intracellular bivalent form of iron that potentiates ART’s efficacy.Table 1IC50 (72h) on different forms of iron in three different MM cell linesMM cell lineIC50 μΜ (RPMI-1640)IC50 μΜ (RPMI+Fe+2)IC50 μΜ (RPMI+Fe+3)IC50 μΜ (RPMI+holotransferin)JJN316.42.7***14.6∼7.9***INA-618.94.1***17.5∼12.8**RPMI-82266.72.3***5.1*3.9*Fe+2 : iron sulfate heptahydrate, [Fe+2]= 0.8mg/L, Fe+3 :iron citrate, [Fe+3]=0.8mg/L, [holotransferin]: 1 μΜ.***p<0.001, **p<0.01, *p<0.05, ∼p<0.1.
Based on the different characteristics of apoptosis induced by ART, we next examined the combined effect of ART with known antimyeloma agents bortezomib, doxorubicin and dexamethasone, that do not share the mechanism of action or the effects of ART on ROS level in the JJN3 cells. ART exhibited an additive or synergistic effect with all agents (Interaction Index≤1.0) implying that low ROS is the aftermath of ART’s molecular action and that through its effect on the mitochondria cannot only act independently but also complement and augment the action of other anti myeloma agents.

Disclosures:
Barlogie: Celgene: Consultancy, Honoraria, Research Funding; Internation Myeloma Foundation: Consultancy, Honoraria; Millennium: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; National Cancer Institute: Research Funding; Johnson & Johnson: Research Funding; Centocor: Research Funding; Onyx: Research Funding; Icon: Research Funding; Myeloma Health, LLC: Patents & Royalties.