Leveraging cytoplasmic nucleosides kinases in AML

Replication of Mitochondrial DNA (mtDNA) requires adequate nucleotide pools from the mitochondria and cytoplasm to support DNA biosynthesis. mtDNA is copied mainly by Mitochondrial DNA Polymerase Gamma (POLG) and replication factors. Additionally, mtDNA biosynthesis is also dependent on nucleotide pools derived from cytoplasmic pathways. Acute Myeloid Leukemia (AML) cells have an increased mitochondrial biosynthesis and reliance on oxidative phosphorylation compared to normal Hematopoietic Cells (HSCs).  

In an article published ahead of print in Blood on 10^th March 2017, Sanduni U. Liyanage and colleagues from the University of Toronto, Canada, discussed their data, which investigated the activity of cytoplasmic nucleotide salvage pathway in AML and its effect on the mtDNA biosynthesis.

The key results of the study were:

• Of human AML samples (n = 542), 55% had higher mtDNA biosynthesis expression compared to normal HSCs (n = 73)
• Compared to normal HSCs, cytoplasmic nucleotide salvage enzymes (phosphorylates nucleosides to nucleotides), Nucleoside Monophosphate Kinase Cytidine/Uridine Monophosphate Kinase 1 (CMPK1) and Nucleoside Diphosphate Kinases (NME1-NME2) were upregulated in AML samples
• Compared to control, knockdown of CMPK1 and Thymidine Kinase 1 (TK1) in OCI-AML2 cells (human AML cell line) reduced mtDNA levels
• Treatment with 2'3'-dideoxycytidine (ddC; a selective inhibitor of POLG that phosphorylates to activated 2'3'-dideoxycytidine triphosphate [ddTCP]) led to an increase in 2'3'-ddTCP in AML samples (n = 6) compared to normal HSCs; P < 0.05
• Treatment of AML cell lines with ddC led to a depletion of mtDNA content and a decrease in protein expression of mtDNA encoded Electron Transport Chain (ETC) subunits COXI and COXII
• Decreased proliferation of AML cells were associated with reduced mitochondrial function and oxidative phosphorylation
• Treatment of SCID mice injected with OCI-AML2 cells with ddC, induced tumor regression and decreased tumor mass compared to control; P < 0.0001
• Treatment of SCID mice injected with patient AML cells with ddC, educed human AML bone marrow engraftment in primary AML (n = 3; P < 0001) and secondary AML (P < 0.01)

In summary, AML cells have elevated cytidine nucleoside kinase pathway activity, which supports mtDNA biosynthesis. Leveraging this “biological vulnerability”, AML cells preferentially activated ddC, which inhibited mtDNA replication, oxidative phosphorylation, and induced anti-AML effects.

Abstract

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 AML samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared to 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 to 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 employed a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated anti-metabolite, 2'3'-dideoxycytidine triphosphate (ddCTP) by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase, POLG. ddC was preferentially activated in AML cells compared to 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 progenitors 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.