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POLG inhibition promotes AML differentiation
Alovudine reduces mtDNA and impairs mitochondrial function in acute myeloid leukemia cells
To investigate the effects of alovudine on mitochondrial function, OCI-AML2 and MV4-11 leukemia cells were treated with increasing concentrations of alovudine. Six days after incubation, changes in mtDNA and bioenerget- ics were measured. Alovudine decreased mtDNA in both OCI-AML2 and MV4-11 cells, although MV4-11 cells were more sensitive with reductions in mtDNA observed at low nM concentrations and >80% reductions in mtDNA observed with 25 nM of the drug (Figure 1B). In
contrast to the large reduction in levels of mtDNA, there was only a small reduction in mitochondrial mass after 6 days of alovudine treatment (Online Supplementary Figure S1) and no change in nuclear DNA (Online Supplementary Figure S2). In keeping with reductions in mtDNA, we noted prominent reductions in protein levels of the mtDNA-encoded respiratory chain IV complex subunits, mt-COXI and mt-COX II (Figure 1C). In contrast, no changes were seen in levels of nu-COX IV, a subunit of the same respiratory chain complex IV, but encoded by nuclear DNA (Figure 1C). mt-COX I and mt-COX II
AB
C
Figure 1. Alovudine inhibits mito- chondrial DNA biosynthesis and oxidative phosphorylation in acute myeloid leukemia (AML). (A) Alovudine’s chemical structure. (B) OCI-AML2 and MV4-11 cells were treated with increasing concentra- tions of alovudine for 6 days. Relative mitochondrial DNA (mtDNA) content was assessed by qRT-PCR as described in the Methods section. Data represent mean+Standard Deviation (SD) mtDNA relative to untreated con- trols from one of three representa- tive experiments. (C) OCI-AML2 and MV411 were treated with increasing concentrations of alovudine. Three and 6 days after treatment, cells were harvested, lysed and levels of cytochrome C oxidase subunits: mitochondrial COXI (mt-COX1), mito- chondrial COXII (mt-COXII), nuclear COX IV (nu-COX IV), and β-tubulin were measured by immunoblotting. The immunoblot from one of three representative experiments is shown. (D) Basal oxygen consump- tion rate (OCR) was assessed in OCI- AML2 and MV411 cells following 6 days of alovudine treatment, using the Seahorse XF96 Metabolic Flux Assay. Data represent the mean±SD basal OCR from one of three repre- sentative experiments. n=6. (E) OCI- AML2 and MV411 cells were treated with increasing concentrations of alovudine for 6 days. ATP production was assessed by CellTitre-Glo Luminescent Cell Viability Assay. Data represent the mean±SD from two independent experiments in triplicate. (F) OCI-AML2 and MV411 cells were treated with increasing concentrations of alovudine. Cell growth and viability was assessed
D
E
F by trypan blue exclusion staining at increasing times after incubation. Data represent the mean±SEM from one of three representative experiments. For all experiments, ***P<0.001 and ****P<0.0001 using Dunnett’s multiple compar- isons test after one-way ANOVA (B, D
and E). (F) Two-way ANOVA.
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