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D. Yehudai et al.
Eukaryotic cells have two separate genomes: nuclear DNA (nuDNA), organized in chromosomes, and the cir- cular mitochondrial DNA (mtDNA), organized within nucleoids located within the mitochondria. Mitochondrial DNA is comprised of a double-stranded circular genome that is 16.6 kb in length and lacks introns. It encodes two rRNAs, 22 t-RNAs, and 13 of the 90 proteins in the mitochondrial respiratory chain, the cascade of enzymes central to ATP production in the mitochondria via oxidative phosphorylation.2 Synthesis of the 13 proteins encoded by the mtDNA occurs in the mitochondrial matrix on mitochondrial ribosomes using mitochondrial-specific protein synthesis machinery.7,8 The remaining approximately 1300 mitochondrial pro- teins are encoded by nuclear genes and translated in the cytoplasm.
Contrary to nuDNA, that replicates once during cell division, mtDNA is continuously replicated independ- ently from the cell cycle.9 Mitochondria contain their own specialized machinery for DNA replication, tran- scription and translation of the mitochondrial genome. Loss of integrity of mtDNA results in dysfunctional res- piratory complexes and negatively affects the production of ATP.
In humans, the nuclear-encoded mitochondrial POLG is the sole polymerase responsible for mitochondrial DNA replication. Human POLG is comprised of a cat- alytic polymerase domain at the C-terminus and an exonuclease domain separated by a linker region at the N-terminus. The holoenzyme consists of the primary subunit POLG and a homodimeric form of its accessory subunit POLG2.10 POLG forms a multi-protein-DNA complex, termed nucleoid, which acts as a hub for mtDNA replication, transcription and translation.11
Mitochondrial and cytoplasmic pathways support mtDNA biosynthesis by supplying it with sufficient nucleotide pools. The former, the mitochondrial nucleotide salvage pathway, converts nucleoside precur- sors to nucleotides by a cascade of kinases within the mitochondria,12 while in the latter, the cytoplasmic path- way, kinases catalyze the phosphorylation of nucleo- sides to nucleotides in the cytoplasm. Nucleosides in this cytoplasmic pathway are synthesized from de novo biosynthesis,13,14 and following their phosphorylation to nucleotides they are imported into the mitochondria by specific nucleotide transporters.15,16
In this current study, we investigated the POLG inhibitor, dideoxynucleoside analog of thymidine, alovu- dine (3'-deoxy-3'-fluorothymidine, FLT), in AML. We evaluated the effects of alovudine on mitochondrial func- tion as well as on stemness and differentiation in AML.
Methods
Cell lines and primary samples
OCI-AML2, MV4-11 and K562 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) augmented with 10% fetal bovine serum (FBS) and antibiotics. TEX cells (a gift from Dr. J. Dick) were cultured in IMDM augmented with 20% FBS, 2 mM L-glutamine, 2 ng/mL human Interleukin-3 (IL-3), 20 ng/mL human stem cell factor (SCF) (R&D Systems) and antibiotics. NB4 cells were cultured in RPMI 1640 medium augmented with 10% FBS. A total of 8227 cells (a gift from Dr. J. Dick) were cultured in X-VIVO 10 supplemented with 20% BIT (StemCell Technologies,
Vancouver, Canada), 10 ng/mL hIL-3, 50 ng/mL hSCF, 10 ng/mL G-CSF, and 25 ng/mL TPO (Pepro Tech).
Peripheral blood was collected from consenting patients with AML. Samples with at least 80% leukemic blasts among low-den- sity cells isolated by Ficoll density gradient centrifugation were included in this analysis. Normal hematopoietic cells were derived from healthy volunteers donating peripheral blood stem cells (PBSCs) for allogeneic stem cell transplantation following granulo- cyte colony-stimulating factor (G-CSF) mobilization. Primary AML and normal hematopoietic mononuclear cells were cultured in Iscove-modified Dulbecco medium (IMDM) supplemented with 20% FBS, 2 mM L-glutamine, 2 ng/mL human IL-3, and 20 ng/mL human SCF. Sample collection and the use of human tissue were approved by the University Health Network institutional review.
All cell lines and primary samples used in our experiments were incubated at 37°C and 5% CO2 in humidified atmosphere.
Xenograft models of human acute myeloid leukemia
For in vivo studies, alovudine was supplied by Medivir AB (Huddinge, Sweden). OCI-AML2 leukemia cells (1x106) were injected subcutaneously into the flanks of severe combined immune deficient (SCID) mice (Ontario Cancer Institute, Toronto, ON, Canada). After the appearance of a palpable tumor (9-11 days), the mice were treated orally with alovudine (50 mg/kg) twice daily or vehicle (saline) control (n=10 per group) at a treat- ment schedule of 5 out of 7 days for a total of 21 days (total num- ber of 27 doses). Tumors were measured 3 times a week based on caliper measurements of tumor length and width (volume=tumor length x width2 x 0.5236). At the end of treatment, mice were sac- rificed and tumor volumes and mass were measured from excised tumors. Mitochondrial DNA (mtDNA) assessment was also car- ried out from excised tumors, using qRT-PCR.
To test alovudine efficacy in a primary AML engraftment mouse model, a frozen aliquot of primary AML cells was thawed, counted, and re-suspended in phosphate-buffered saline. Viable trypan blue-negative cells (2.5x106) were injected into the right femur of 10-week old female NOD-SCID mice that were sub- lethally irradiated (2 Gy), and pretreated with 200 μg of anti- mouse CD-122. Mice were treated once daily with oral alovudine at 25 mg/kg or vehicle (saline) control (n=10 per group) 5 out of 7 days for 24 days (total number of 17 doses). Mice were then sac- rificed, femurs flushed, and primary AML engraftment (CD45+CD33+CD19- cells) in the left femur was determined by flow cytometry.
In vivo studies were performed according to the regulations of the Canadian Council on Animal Care and with the approval of the Ontario Cancer Institute Animal Ethics Review Board.
Results
The nucleoside analog alovudine depletes mitochondrial DNA and reduces cell growth and viability in acute myeloid leukemia cells
Alovudine (Figure 1A) is a dideoxynucleoside analog of thymidine, originally developed as a reverse transcriptase inhibitor and evaluated in patients for the treatment of HIV.17-19 In addition to inhibiting viral replication, alovu- dine triphosphate is incorporated into DNA by POLG preferentially over nuclear polymerases, resulting in chain termination and inhibition of its enzymatic activity in cell- free assays.20 However, its anti-leukemic activity has not been previously described. Therefore, we focused our fur- ther investigations on this compound.
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haematologica | 2019; 104(5)