Here we report that mefloquine, a quinoline approved for the treatment and prevention of malaria (23
), has toxicity for human AML cells including AML progenitors, while sparing normal human hematopoietic cells treated with the same doses. Furthermore, we show that the antileukemic effects of mefloquine are mediated through disruption of lysosomes, a previously unappreciated mechanism of action of this drug.
In the treatment of mild-moderate Plasmodium falciparum
malaria, patients receive a single oral dose of up to 1,250 mg mefloquine, which is usually sufficient to eradicate the parasite (24
). However, in regions where mefloquine-resistant malaria is prevalent, patients have received higher doses, up to 25 mg/kg, of drug without significant toxicity (58
). In addition, serum concentrations of mefloquine up to 5 μM have been reported in individuals receiving 250 mg weekly for malaria prophylaxis (59
). Thus, antileukemia concentrations of mefloquine may be pharmacologically achievable.
Although mefloquine is known to accumulate in the lysosomes of the malarial parasite (42
), its mechanism of action as an antimalarial is unclear. Therefore, this work may also inform about the mechanism of action of mefloquine in malaria, as it may also selectively disrupt the lysosomes of the malarial parasite. While we demonstrated that mefloquine disrupts lysosomes and our yeast study strongly suggests that an intact lysosome is required for resistance to mefloquine, future studies to identify its direct binding target(s) in the lysosomes of leukemia cells and possibly the malarial vacuole will be of major interest.
Autophagy is a lysosome-mediated process of cellular autodigestion that, in times of nutrient deprivation, enables the cell to recycle raw materials for macromolecular and organellar biosynthesis. Both inhibiting and promoting autophagy can lead to cell death in cancer (ref. 61
and reviewed in ref. 62
). The antimalarial chloroquine is structurally similar to mefloquine and inhibits the degradation of autophagy targets in the autophagolysosome. Through this mechanism, chloroquine can induce cell death and sensitize cells to chemotherapy (including imatinib mesylate in chronic myeloid leukemia; refs. 63
) and radiation. However, as it involves induction of lysosome disruption, the mechanism of action of mefloquine appears distinct from that of chloroquine and other inhibitors of autophagy.
LAMP1 and LAMP2 are the predominant proteins in the lysosomal membrane, accounting for approximately 50% of the protein mass contained therein (14
). Previous studies have demonstrated that knockdown of LAMP1 or LAMP2 disrupts the integrity of lysosomes in malignant cells but not in non-transformed cells (50
). Consistent with this finding, we observed that knockdown of LAMP proteins decreases the proliferation of leukemic cells, and also decreases lysosome integrity.
Our results are also in agreement with prior reports showing increased cathepsin expression (67
) and lysosomal mass (50
) upon oncogene-mediated transformation. Prior studies indicated that increased lysosomal biogenesis can lead to increases in lysosomal volume (54
). In addition, Ono et al. (68
) previously suggested that an increased lysosomal volume would render cells more sensitive to death after lysosome disruption with blue light irradiation, although the mechanism was unclear. These findings taken together with those we now report provide a strong rationale for therapeutically targeting the lysosome in AML, as larger lysosomes may be more sensitive to disruption. Further support for this idea comes from our results indicating that the cells in mefloquine-sensitive AML samples also had a significantly increased lysosomal mass, as measured by LysoTracker dye uptake, as compared with cells in either mefloquine-insensitive samples or normal hematopoietic sources. Thus, increased lysosomal size or mass may be a predictive biomarker of sensitivity to lysosomal disruption in human AML.
Currently, it is unclear why AML cells have increased lysosomal mass and biogenesis. Given the role of lysosomes in macromolecule and organelle recycling, they may function to increase levels of amino acids and nucleotides necessary for cell proliferation. Indeed, a recent report indicated that intracellular levels of amino acids can regulate the expression of TFEB, a transcription factor responsible for activation of lysosomal biogenesis pathways (54
). In addition, increased lysosomal mass in AML may be necessary to provide a source of fatty acids for these cells. Recently, it was demonstrated that AML cells have higher reliance on fatty acids for their energy supply, and AML cells and their most primitive elements have increased rates of fatty acid oxidation (72
In summary, a combined approach using small-molecule and yeast mutant screens with in vitro and in vivo functional studies of AML has identified lysosome disruption as a novel and promising therapeutic strategy for human AML. Investigation of the mechanistic basis of the selective sensitivity of primary human AML cells to agents that disrupt lysosomes revealed increased lysosomal size and biogenesis in bulk AML cells and subsets enriched in AML stem cells. Thus, in spite of the genetic and biological heterogeneity of AML, some common biological features may remain accessible to selective targeting and potential therapeutic exploitation.