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D. Yehudai et al.
of the primary subunit POLG and a homodimeric form of its accessory subunit POLG2.10 The nuclear-encoded pri- mary subunit contains a C-terminal catalytic polymerase domain and N-terminal exonuclease domain separated by a linker/spacer region.36 The polymerase domain is responsible for extension of the DNA strand, and similar to nuclear-DNA polymerase, utilizes deoxynucleotide triphosphates (dNTPs) as its substrate to the growing chain.37 Any alteration of dNTP (i.e. analogs of dNTPs, called ddNTPs) can inhibit POLG function through chain termination. We showed that alovudine decreased the growth of OCI-AML2 cells xenografted into SCID mice.
However, in sublethally irradiated NOD/SCID mice engrafted by intrafemural injection with a primary AML patient sample, alovudine produced only a small reduction in primary AML engraftment. Unfortunately, in this mouse model, the maximum tolerated dose of alovudine was only 25 mg/kg/day. In contrast, in SCID mice engraft- ed with OCI-AML2 cells, the maximum tolerated dose was over 50 mg/kg/day. We suspect that the difference in the maximal tolerated dose is due to differences in the mouse strains or the sublethal irradiation that the NOD/SCID mice received prior to engraftment with pri- mary AML cells. Alternatively, the reduced efficacy in the
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Figure 6. Polymerase gamma (POLG) knockdown promotes acute myeloid leukemia differentiation. OCI-AML2 cells were infected with shRNA targeting POLG or a control sequenced in lentiviral vectors. (A) POLG knockdown at 14 days post transduction was assessed by qRT-PCR. Data represent the mean+Standard Deviation (SD) mtDNA from one of three representative experiments. (B) Relative mitochondrial DNA (mtDNA) content was assessed by qRT-PCR 14 days post transduction with POLG shRNA, as described in the “Methods” section. Data represent the mean+Standard Error of Mean (SEM) from two independent experiments. (C) Cells were harvested 11, 14 and 18 days post POLG knockdown and levels of cytochrome C oxidase subunits: mitochondrial COXI (mt-COX1), nuclear COX IV (nu-COX IV), and β-tubulin in whole-cell extracts were measured by immunoblotting. The immunoblot from one of three representative experiments is shown. (D) Basal oxygen con- sumption rate (OCR) was assessed 10 days following POLG knockdown, using the Seahorse XF96 Metabolic Flux Assay. Data represent Mean±SEM basal OCR from two independent experiments; n=6. (E) Cell growth and viability were assessed by trypan blue exclusion staining. Data represent the mean±SD from one of three representative experiments. (F) CD11b expression was assessed by flow cytometry. Data represent the mean±SD from one of three representative experiments. For all experiments, *P<0.5, **P<0.01, ***P<0.001, and ****P<0.0001 using Dunnett's multiple comparison following one-way ANOVA (A and F) or two-way ANOVA (B and D). (E) Two-way ANOVA test.
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haematologica | 2019; 104(5)