B-cell acute lymphoblastic leukemia (ALL) is derived from an accumulation of malignant, immature B cells in the bone marrow and blood. Relapse due, in part, to the emergence of tumor cells that are resistant to front line standard chemotherapy is associated with poor patient outcomes. This challenge highlights the need for new treatment strategies to eliminate residual chemoresistant tumor cells. Based on the use of pitavastatin in acute myeloid leukemia (AML), we evaluated its efficacy in an REH ALL cell line derived to be resistant to vincristine. We found that pitavastatin inhibited the proliferation of both parental and vincristine-resistant REH tumor cells at an IC₅₀ of 449 nM and 217 nM, respectively. Mitochondrial bioenergetic assays demonstrated that neither vincristine resistance nor pitavastatin treatment affected cellular oxidative phosphorylation, beta-oxidation, or glycolytic metabolism in ALL cells. In a co-culture model of ALL cells with bone marrow stromal cells, pitavastatin significantly decreased cell viability more robustly in the vincristine-resistant ALL cells compared with their parental controls. Subsequently, NSG mice were used to develop an in vivo model of B-cell ALL using both parental and vincristine-resistant ALL cells. Pitavastatin (10 mg/kg i.p.) significantly reduced the number of human CD45+ REH ALL cells in the bone marrow of mice after 4 weeks of treatment. Mechanistic studies showed that pitavastatin treatment in the vincristine-resistant cells led to apoptosis, with increased levels of cleaved PARP and protein-signaling changes for AMP-activated protein kinase/FoxO3a/Puma. Our data suggest the possible repurposing of pitavastatin as a chemotherapeutic agent in a model of vincristine-resistant B-cell ALL.

The lack of complete therapeutic success in the treatment of B-cell acute lymphoblastic leukemia (ALL) has been attributed, in part, to a subset of cells within the bone marrow microenvironment that are drug resistant. Recently, the cholesterol synthesis inhibitor, pitavastatin (PIT), was shown to be active in acute myeloid leukemia, prompting us to evaluate it in our in vitro co-culture model, which supports a chemo-resistant ALL population. We used phospho-protein profiling to evaluate the use of lipid metabolic active compounds in these chemo-resistant cells, due to the up-regulation of multiple active survival signals. In a co-culture with stromal cells, a shift towards anabolic processes occurred, which was further confirmed by assays showing increased lipid content. The treatment of REH leukemia cells with pitavastatin in the co-culture model resulted in significantly higher leukemic cell death than exposure to the standard-of-care chemotherapeutic agent, cytarabine (Ara-C). Our data demonstrates the use of pitavastatin as a possible alternative treatment strategy to improve patient outcomes in chemo-resistant, relapsed ALL.

Reprogramming of cellular pathways is a crucial mechanism of drug resistance and survival in refractory acute lymphoblastic leukemia (ALL) cells. In the present study, we performed an unbiased gene expression analysis and identified a dysfunctional mitochondrial respiration program in drug-resistant ALL cells grown in a co-culture system with bone marrow stromal cells (BMSC). Specifically, the activity of the complexes within the electron transport chain was significantly downregulated, correlated with decreased mitochondrial mass and ATP production in drug-resistant ALL cells. To validate mitochondrial respiration as a druggable target, we utilized pyrvinium pamoate (PP), a known inhibitor of mitochondrial respiration and documented its anti-leukemic activity in several ALL cell lines grown alone or in co-culture with BMSC. To increase the bioavailability profile of PP, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. PP anti-leukemic activity was decreased by the addition of sodium pyruvate, and furthermore, PP was found to have an additive anti-leukemic effect when used in combination with rotenone, a mitochondrial complex I inhibitor with activity similar to PP on the mitochondrial respiration. Importantly, PP’s cell death activity was found to be specific for leukemic cells as primary normal immune cells were resistant to PP-mediated cell death. In conclusion, we have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells.