Halofuginone, an Old Anticoccidosis Drug, Induces Apoptosisin Multiple Myeloma Cells, Associated with Down Regulation of MCL1.

Halofuginone, a synthetic derivative of quinazolinone alkaloid, has recently been shown to have an anti-cancer effect in various solid and hematological malignancies. It is an orally available drug with a safe toxicity profile in clinical trials of inflammatory disease and has anti-angiogenic, anti-metastatic and anti-proliferative effects in preclinical studies. It blocks TGFb signaling by targeting Smad 2/3, inhibits NFkB activity, as well as downregulates extra cellular matrix (ECM) formation via the inhibition of collagen type I formation and matrix metalloproteinase 2(MMP2). Since ECM has an important role in the bone marrow microenvironment in multiple myeloma (MM) pathogenesis, we here examined whether halofuginone induces cytotoxicity against various MM cell lines, including those sensitive and resistant to conventional and novel chemotherapies. Halofuginone (12.5–400 nM) induced cytotoxicity in a dose-dependent fashion in a variety of MM cell lines (n=20), regardless of the sensitivity to conventional chemotherapy (i.e., dexamethasone, melphalan, doxorubicin) and novel therapies (i.e., lenalidomide, bortezomib) with IC50 of 50–200 nM. In contrast, halofuginone did not induce cytotoxicity against CD40-activated peripheral blood mononuclear cells from normal donors, even at high doses (500–1000nM). Importantly, neither IL6 nor IGF1, two key growth and survival factors in MM, overcame the growth inhibitory effect of halofuginone. We further examined molecular mechanisms whereby halofuginone triggers growth inhibition in MM cells. It induced both apoptosis and necrosis in a time- (2–48h) and dose (50–200 nM) dependent manner in MM1R and OPM1 MM cells, assessed by annexin V/ PI staining. Significant dose- dependent activation of caspase 3 and 8 was demonstrated after 25–200 nM of halofuginone treatment of MM1R, MM1S, and OPM1 cells. Mitochondrial membrane potential was also reduced after 24 hours of halofuginone treatment (50, 200 nM) of MM1R and OPMI cells, as evident by the accumulation of JC1 monomers. In addition, western blot analysis showed that halofuginone induces cleavage of apoptotic proteins caspase 3/8 and PARP, and downregulates anti-apoptotic protein MCL1. Taken together, these data suggest that halofuginone has significant anti-MM activities and is a potential novel therapy in MM.