Acute myeloid leukemia (AML) is an aggressive, genetically heterogeneous malignancy wherein immature progenitors crowd out the normal hematopoietic cells, leading to marrow failure and death. In 2013, it is projected that there will be 13,780 new cases and 10,200 deaths from AML in the United States. Even with decades of research into this disease, treatments remain unchanged and patient survival dismal. Standard treatment for AML is combination chemotherapy with cytarabine and an anthracycline. Treatment will often lead to a remission; however, most patients relapse with chemoresistant disease. This highlights the need for the development of new therapies. Ribonucleotide reductase (RR) facilitates the rate-limiting reaction in DNA synthesis. Hydroxyurea (HU) is a well-known inhibitor of RR, resulting in the depletion of purine nucleotides. It has a role in AML as a cytoreductive agent and in the palliative setting, where other regimens have been deemed too intensive. However, its myelosuppressive and GI toxicities are dose-limiting. Didox is an inhibitor of RR, which inhibits both purine and pyrimidine synthesis, and has been shown to be less myelosuppressive than HU.
In our previous studies in a panel of human AML cell lines, Didox had an average IC50 of 5.76 μM (range 3.62–7.37 μM). To evaluate if Didox treatment resulted in DNA damage we assessed γH2AX foci formation in KG1a cells. After a 24-hour exposure to a titration of Didox nuclei were scored for the presence of γH2AX foci. Didox induced DNA damage in this erythroleukemia cell line at 24 hours, with a mean score of 0.480 in the negative control, 0.852 with 5 μM, 0.907 with 10 μM, and 0.976 at 20 μM Didox (p=0.0015 by 1-way ANOVA). Consistent with Didox exposure resulting in DNA damage, we saw induction of p53 in OCI/AML3 cells by Western blot following Didox treatment. To determine if Didox exposure culminated in induction of apoptosis, we performed an annexin V/propidium iodide assay. Didox induced apoptosis in KG1a cells (45.27% positive compared to 3.15% positivity in the negative control). To determine the effect of Didox on leukemic progenitor cells we exposed KG1a cells to a titration of Didox for 24 hours and then performed a colony formation assay. Didox reduced the colony-forming potential of KG1a cells (5% at 200nM, 21.9% at 2μM, 60.6% at 20 μM, and 84.3% reduction at 200 μM). These data demonstrate that Didox is an active agent which inhibits RR resulting in DNA damage, p53 induction and culminating in cell death via apoptosis.
Previous in vivo studies carried out in syngeneic, therapy-resistant MLL-ENL-driven AML models (one expressing Flt3 ITD and one over-expressing NRasG12D) yielded a reduction in leukemic burden (p= 0.0342 and 0.0026, respectively) and a survival benefit (p= 0.0094 and 0.0001, respectively) with a five-day Didox treatment at 425 mg/kg/d compared to vehicle-treated controls. We evaluated the impact of Didox given in this fashion on normal progenitors by treating normal 8-week-old C57Bl/6. 72 hours post-treatment, the animals were sacrificed and the bone marrow, sternum, and small intestine (SI) were harvested for toxicity studies. The sternum and SI from the Didox and control-treated samples were reviewed by a veterinary pathologist blinded to the treatment group. No differences were observed between Didox treated and control groups. To determine if Didox exposure affected the activity of normal hematopoietic stem cells bone marrow was harvested from treated mice and injected into Ly5.1+ secondary recipients. After 21 days recipient mice were sacrificed, bone marrow harvested, and evaluated for engraftment. Preliminary data suggested that Didox treatment did not alter engraftment potential of normal progenitors (Didox treated bone marrow 87.44% versus control marrow 89.75% engraftment).
Didox is a RR inhibitor with activity in models of AML. Our in vitro data indicate that Didox is active against leukemic progenitor cells with reduced colony-formation potential of treated cells. Furthermore, at the effective dose, Didox was not toxic to normal tissues. Most importantly, preliminary data suggests it does not alter the engraftment potential of normal hematopoietic cells. The activity of Didox combined with its favorable toxicity profile makes it an ideal candidate for further translation to the clinic.
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