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POLG inhibition promotes AML differentiation
ABC
D
E
Figure 5. Effects of inhibiting mitochondrial translation and polymerase gamma (POLG) on acute myeloid leukemia growth and differentiation. (A-C) OCI-AML2 cells were treated with increasing concentrations of Tigecycline for 72 hours (h). (A) Cell growth and viability was assessed by Alamar Blue assay [Mean+Standard Deviation (SD)]. (B) Cells were harvested 72 h after treatment, lysed, and levels of cytochrome C oxidase subunits: mitochondrial COXI (mt-COXI), mitochondrial COXII (mt-COXII), nuclear COX IV (nu-COX IV), and β-tubulin measured by immunoblotting. The immunoblot from a representative experiment is shown. (C) CD11b expres- sion was assessed by flow cytometry. (D) OCI-AML2 cells were treated with increasing concentrations of antiviral ddC. Cell growth and viability was assessed by trypan blue exclusion staining at increasing times after incubation. Data represent the mean±Standard Error of Mean (SEM) from one of three representative experiments. (E) OCI-AML2 cells were treated with increasing concentrations of 2'3'-dideoxycytidine (ddC) for 10 days. CD11b expression was assessed by flow cytometry. For all experiments, *P<0.5, **P<0.01, ***P<0.001, and ****P<0.0001 using Dunnett's multiple comparisons test after one-way ANOVA (C) and two-way ANOVA (D). (E) Unpaired t-test.
ing levels of mitochondrial encoded respiratory chain pro- teins and basal oxygen consumption without altering lev- els of mtDNA would be sufficient to mimic the effects of alovudine and induce AML differentiation. We treated OCI-AML2 cells with tigecycline that we previously showed inhibits mitochondrial protein translation by tar- geting mitochondrial ribosomes. Consistent with our pre- vious studies,4 tigecycline reduced levels of mtDNA- encoded proteins but not nuclear-DNA-encoded proteins and reduced the proliferation and viability of AML cells (Figure 5A and B). However, there was no evidence of dif- ferentiation (Figure 5C).
Then, we asked whether other chemical or genetic inhibitors of POLG would also promote AML differentia- tion. We treated OCI-AML2 cells with the antiviral 2'3'-dideoxycytidine (ddC) that we and others previously showed inhibited POLG.6,20 Similar to alovudine, ddC reduced mtDNA and reduced levels of mitochondrial encoded respiratory chain proteins.6 Like alovudine, ddC also reduced AML growth (Figure 5D) and promoted AML differentiation (Figure 5E). As a genetic approach, we knocked down POLG with shRNA in lentiviral vec- tors. Partial POLG knockdown reduced mtDNA, but the reductions in mtDNA were not sufficient to significantly reduce levels of respiratory chain proteins or impair oxidative phosphorylation. However, partial POLG knockdown mimicked the effects of alovudine and reduced AML proliferation, and induced differentiation of AML cells (Figure 6A-F).
Thus, taken together, our data suggest that inhibition of POLG promotes AML differentiation through effects that
are independent of reductions in oxidative phosphoryla- tion or respiratory chain proteins.
Discussion
Inhibiting nuclear DNA replication by targeting nuclear
DNA polymerases is the primary mechanism of many
standard anti-leukemic agents, including cytarabine, clo-
farabine and cladribine. Some nucleoside analogs also
cross react with the mitochondrial polymerase, POLG,
and thereby inhibit mitochondrial DNA replication. Given
that a subset of AML cells and stem cells have increased
mitochondrial mass, mitochondrial DNA, and a greater
reliance on oxidative phosphorylation,3,5,29 we sought to
identify new nucleoside analogs that deplete mitochondr-
ial DNA. In this current study, we evaluated alovudine as
a potent nucleoside analog targeting mtDNA in AML.
Alovudine is a dideoxynucleoside analog of thymidine,
which inhibits mtDNA polymerase activity through its 5’-
triphosphate metabolite. First introduced in the early 90s,
this drug was originally developed as a reverse transcrip-
tase inhibitor and evaluated in patients for the treatment
of HIV.30-32 More recently, it has been labeled with 18F and
used as a reagent in PET imaging to visualize malignan- cy.33-35
We demonstrated that inhibition of POLG with alovu- dine inhibits the viability and proliferation of AML cells in vitro and in vivo. Human mitochondrial DNA is replicat- ed exclusively by the mitochondrial DNA polymerase gamma. The holoenzyme is a heterotrimer which consists
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