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POLG inhibition promotes AML differentiation
est expression of POLG (Online Supplementary Figure S4D). There was no increase in apoptosis as measured by Annexin V staining 6 days after alovudine treatment, a time at which reductions in cell viability and mtDNA biosynthesis were already evident. However, cell death was observed after more prolonged (11-day) incubation with alovudine (Online Supplementary Figure S4E). Furthermore, analysis of the cell cycle in alovudine-treated AML cells showed little or no change (Online
Supplementary Figure S5).
Next, we investigated the effects of alovudine on pri-
mary AML cells and normal hematopoietic cells. Primary AML and normal hematopoietic cells were treated for 6 days with increasing concentrations of alovudine. After incubation, we measured cell viability and levels of mtDNA. The number of viable primary cells treated with the vehicle control did not increase during the period of incubation, suggesting that the primary cells were quies- cent and did not proliferate. Alovudine reduced levels of mtDNA in primary AML cells at concentrations that also reduced cell viability (Figure 2A and B and Online Supplementary Table S1). In contrast, alovudine did not reduce the viability of the normal hematopoietic cells (Figure 2B). Furthermore, alovudine reduced the clono- genic growth of primary AML cells. We also tested the effects of alovudine on the clonogeneic growth of normal hematopoietic cells. Compared to AML cells, normal hematopoietic cells were more resistant to alovudine. However, at the highest tested dose (2000 nM), alovudine partially reduced the clonogenic growth of granulocyte precursors (CFU-GM) and completely inhibited the growth of primitive erythroid progenitor cells (BFU-E) (Figure 2C and D and Online Supplementary Table S1).
Thus, alovudine inhibits mtDNA biosynthesis in pri- mary AML cells and selectively targets a subset of AML cells in vitro.
Alovudine displays efficacy in mouse models of human leukemia
Given the anti-leukemic effects of alovudine in vitro, we examined the efficacy and toxicity of alovudine in a mouse model of leukemia. Severe combined immune deficient (SCID) mice xenografted with OCI-AML2 cells were treat- ed with alovudine (50 mg/kg bid) or vehicle control by oral gavage. Alovudine reduced the growth of leukemia in vivo by approximately 70% without evidence of toxicity (Figure 3A). Specifically, doses of alovudine that reduced tumor growth did not alter mouse body weight, behavior, serum chemistries, or organ histology (Figure 3B and Online Supplementary Figure S6). Finally, we conducted correlative studies and measured mtDNA in leukemic cells isolated from mice treated with alovudine. We observed reductions in mtDNA by greater than 75% in OCI-AML2 tumors excised from mice treated with alovudine (Figure 3C).
Next, we tested whether alovudine targets primary AML cells in vivo. Sublethally irradiated NOD-SCID mice preconditioned with anti-CD122 received intrafemural injection of primary AML cells. In this mouse model, the maximum tolerated dose of alovudine was only 25 mg/kg/day and less than the dose tolerated by the SCID mice engrafted with OCI-AML2 cells. Therefore, mice were treated with 25 mg/kg alovudine for 5 of 7 days for 17 days. A small, but statistically significant reduction in primary AML engraftment was observed (Online Supplementary Figure S7), potentially reflecting the reduced
treatment dose, an alovudine-resistant primary AML sam- ple, or a protective effect from the marrow niche.
Alovudine promotes monocytic differentiation in acute myeloid leukemia
Recent studies reported that mitochondrial pathways and metabolism can regulate the differentiation of malig- nant cells.21-24 Therefore, we explored the effects of alovu- dine on the differentiation of AML cells. OCI-AML2 and MV4-11 cells were treated with concentrations of alovu- dine that depleted mtDNA for 10 days. Alovudine increased expression of CD11b, a cell surface marker asso- ciated with monocytic differentiation (Figure 4A). It also induced morphological changes typical of monocytic dif-
Figure 3. Alovudine displays efficacy in mouse models of human acute myeloid leukemia. OCI-AML2 cells were injected subcutaneously into the flank of SCID mice. Once tumors were palpable, mice were treated with oral (PO) alovudine (50 mg/kg bid) or vehicle control for 14 days. (A) Tumor volume was assessed from excised tumors. The error bars represent Standard Deviation (SD) (n=10 per group). (B) Body weight was assessed every 2-3 days. The error bars repre- sent SD (n=10 per group). (C) Relative mitochondrial DNA (mtDNA) from xenograft tumors excised in (B) (n=5 per group). For all experiments, ***P<0.001 and ****P<0.0001 using two-way ANOVA (A and B) and t-test (C).
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