Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.

The thymidine dideoxynucleoside analog, alovudine, inhibits the mitochondrial DNA polymerase γ, impairs oxidative phosphorylation and promotes monocytic differentiation in acute myeloid leukemia.

Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.

AML refractory to primary induction with Ida-FLAG has a poor clinical outcome.

We evaluated outcomes of 100 patients with high risk AML treated with Ida-FLAG induction as first-line therapy. 72 achieved remission with one cycle; 19 did not. High risk cytogenetics and TP53 mutations were associated with failure to achieve remission. In those reaching remission, allogeneic bone marrow transplantation was associated with better relapse-free and overall survival. Those not achieving remission with induction therapy were extremely unlikely to reach remission with further therapy and had a dismal prognosis. Exploratory molecular analysis confirmed persistence of the dominant genetic mutations identified at diagnosis. Ex vivo chemosensitivity did not demonstrate significant differences between responders and non-responders. Thus, Ida-FLAG induction has a high chance of inducing remission in patients with high risk AML. Those achieving remission require allogeneic transplantation to achieve cure; those not achieving remission rarely respond to salvage chemotherapy and have a dismal outcome. Alternatives to conventional chemotherapy must be considered in this group.

Characterizing the mitochondrial DNA polymerase gamma interactome by BioID identifies Ruvbl2 localizes to the mitochondria.

Human mitochondrial DNA (mtDNA) is replicated by the mitochondrial DNA polymerase gamma (POLG). Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new interaction partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this protein. Ruvbl2 was detected in mitochondria-enriched fractions in leukemic cells. Additionally, transgenic overexpression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a potential novel mitochondrial isoform of Ruvbl2.

Mitochondrial Targeting of Doxorubicin Eliminates Nuclear Effects Associated with Cardiotoxicity.

The highly effective anticancer agent doxorubicin (Dox) is a frontline drug used to treat a number of cancers. While Dox has a high level of activity against cancer cells, its clinical use is often complicated by dose-limiting cardiotoxicity. While this side effect has been linked to the drug's direct activity in the mitochondria of cardiac cells, recent studies have shown that these result primarily from downstream effects of nuclear DNA damage. Our lab has developed a mitochondrially targeted derivative of Dox that enables the selective study of toxicity generated by the presence of Dox in the mitochondria of human cells. We demonstrate that mitochondria-targeted doxorubicin (mtDox) lacks any direct nuclear effects in H9c2 rat cardiomyocytes, and that these cells are able to undergo mitochondrial biogenesis. This recovery response compensates for the mitotoxic effects of Dox and prevents cell death in cardiomyocytes. Furthermore, cardiac toxicity was only observed in Dox but not mtDox treated mice. This study supports the hypothesis that mitochondrial damage is not the main source of the cardiotoxic effects of Dox.

Targeting mitochondrial RNA polymerase in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.