Nanoparticle Arsenic Compound Realgar Effectively Targets Myeloma Stem-Like Side Population
Authors: Jakubikova Jana , Hideshima Teru , Groen Richard W.J. , Cholujova Danka , Bujnakova Zdenka , Laubach Jacob P. , Munshi Nikhil C. , Richardson Paul G. , Mitsiades Constantine S. , Balaz Peter , Sedlak Jan , Anderson Kenneth C. 
Published: October 2019

Introduction
Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy.
Arsenic trioxide (ATO, As2O3) shows only limited responses compared to anti-MM agents such as bortezomib and lenalidomide in relapsed and refractory MM. Here we evaluated the anti-MM activity of another arsenic compound (arsenic sulfide, As4S4), used in traditional Chinese medicine with greater efficacy and less genotoxicity than ATO, prepared by milling into realgar nanoparticles (NREA). We assessed the effect of NREA on MM stem cell-like SP fraction in the context of the bone marrow stromal cells (BMSCs) to provide the rational for its clinical evaluation.

Methods and Results
First, we evaluated the effect of both NREA and ATO on MM cell survival. Both drugs significantly decreased survival of 13 MM cell lines in a concentration-dependent manner. Importantly, we observed more potent anti-MM effect with NREA compared to ATO, with IC50 values of NREA 2-4 times lower than ATO. Moreover, we confirmed the effect of NREA on MM cells from patients with primary relapsed/refractory disease: NREA showed a dose-dependent response, with IC50 values in the range 1-2 μM. Significant concentration-dependent increased apoptosis was observed in NREA-treated MM cells. Similarly, decreased mitochondrial membrane potential was more significantly triggered by NREA than ATO. Consequently, NREA modulated the MM cell cycle profile: concentration-dependent treatment induced either G0/G1 (MM.1S and KMS11) or G2/M arrest (RPMI-S and OPM1) at 48h. To compare in vivo anti-tumor activity of NREA and ATO, we used our xenograft murine model of human MM. Tumor-bearing mice were intraperitoneally treated with NREA, ATO or with the respective vehicle. Treatment with NREA triggered more significant tumor growth inhibition compared to ATO at day 8 of treatment.
To evaluate the effect of NREA on the SP phenotype of MM cells, we treated MM cells with subtoxic concentrations of NREA and ATO for 72 h. Analysis of MM cells for low intracellular content of Hoechst 33342 (SP fraction) revealed that NREA significantly decreased the percentage of SP cells in MM cells in a dose-dependent manner, whereas increase in SP fraction was observed in ATO-treated cells. To evaluate the effect of the bone marrow microenvironment on targeting the SP cells with NREA, MM cells were cultured alone or with BMSCs, and then analyzed for low intracellular accumulation of Hoechst 33342. Treatment by NREA, but not ATO, significantly depleted SP cells in co-cultures MM cells with BMSCs. Moreover, NREA decreased clonogenic potential, whereas ATO had no effect. Similarly, tumorigenic potential of SP cells was significantly decreased by NREA, but not by ATO, treatment.

Conclusions
Overall, our results demonstrate significant anti-tumor activity of NREA against MM cells in vitro. Our in vivo data further show that NREA significantly decreased tumor burden at clinically achievable concentrations of arsenic. Importantly, targeting of SP cells by NREA in the context of BMSCs provides the preclinical rationale for its clinical evaluation.

Disclosures:
Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.