Artemisinins Synergize with BCL2 Inhibitors By Reducing MCL1 Protein Levels in Leukemia Cells

Artemisinins have broad antineoplastic activity in vitro , are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Both artesunate (AS; the current World Health Organization-recommended drug for severe malaria) and artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a more potent derivative) substantially inhibited acute leukemia growth in vivo and in vitro , at doses where normal CD34⁺ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2015, PMID: 26771236).

To investigate integration of artemisinins into the treatment of acute leukemias, we sought to determine, via a focused drug combination screen, if AS and ART838 could be paired synergistically with emerging antileukemic drugs. Navitoclax (ABT737) and venetoclax (ABT199), the only BCL2 inhibitors in the drug combination screen library of 111 emerging antineoplastic compounds, were 2 of the top 3 candidates identified, synergizing with both AS and ART838 against all 3 acute leukemia cell lines screened. Independent in vitro and in vivo experiments validated the screening results that AS and ART838 synergized strongly with BCL2 antagonists.

Neither the detailed mechanism(s) of action nor the key molecular target(s) of artemisinins are known definitively, in cancers or microbes. Artemisinin activity seems to be mediated predominantly by generation of reduced oxygen species (ROS) by the endoperoxide pharmacophore(s) common to all artemisinins, leading to cell cycle arrest and apoptosis in cancer cell lines. Effects of artemisinins on levels of several pro-apopototic and anti-apoptotic molecules in cancer cell lines have been reported, including reduction of the BCL2 family member MCL1. MCL1 is frequently elevated in primary acute leukemias at initial diagnosis or at relapse, and MCL1 has been demonstrated as a mediator of resistance to BCL2 inhibitors in many AML and ALL cell lines. Therefore, we hypothesized that AS and ART838 might synergize with BCL2 inhibitors against acute leukemias by reducing the intracellular level and/or activity of MCL1.

Levels of MCL1 protein, but not mRNA, were reduced in acute leukemia cell lines treated with AS or ART838. Artemisinin-mediated MCL1 reduction was selective, in that protein levels of other BCL2 family members were not affected by AS or ART838 treatment. When leukemia cells were treated with deoxy-ART838, an analog lacking the endoperoxide pharmacophore and inactive against either leukemias or malaria, caused much less MCL1 reduction. Co-treatment with AS or ART838 plus N-acetyl-cysteine (NAC) similarly decreased the reduction in levels of MCL1 protein. Thus, ROS appears necessary for the artemisinin-mediated post-transcriptional reduction of MCL1 protein levels.

To confirm the potential role of MCL1 reduction in the observed antileukemic efficacy of artemisinins and in the observed synergy of artemisinins with ABT199, we used 3 established selective inhibitors of MCL1 as pharmacologic tools to cause loss-of-MCL1-function. S63845 and A1210477 selectively inhibit binding of BIM to MCL1, whereas maritoclax reduces cellular MCL1 levels by increasing degradation of MCL1 protein. We found that each of these 3 structurally dissimilar compounds added to the antileukemic efficacy of ABT199, but not AS, in AML and ALL cell lines, confirming the potential role for MCL1 reduction in the observed antileukemic efficacy of artemisinins and in the observed synergy of artemisinins with ABT199. Based on our observations, we propose the combination of AS or ART838 plus a BCL2 inhibitor such as ABT199 as a low-toxicity drug pair for incorporation into acute leukemia therapeutic regimens.