The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML.

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML

Primary patient sample characteristics.

<p>Primary patient sample characteristics.</p

Didox is well tolerated.

<p>A. Didox treated C57Bl/6 mice showed no difference in tissue morphology compared to vehicle treated controls as read by a veterinary pathologist blinded to treatment assignment. Representative H&E sections of gastrointestinal tract (Small Intestine) and bone marrow from Didox (n = 3) and control treated animals (n = 3). B. Colony formation assays performed on normal HSCs following 24 hour Didox exposure (0–200 µM), p = 0.09. C. Didox treatment does not harm normal HSCs. C57Bl/6 mice were treated for 5 days with 425 mg/kg Didox or a vehicle control via IP injection. 72 hours post treatment the animals were sacrificed and their marrow harvested. Marrow was then transplanted into lethally irradiated (8 Gy) Ly5.1+ recipients and allowed to engraft. Post-engraftment the animals were sacrificed, marrow harvested, and analyzed for Ly5.2+ by flow cytometry. Engraftment values were normalized to vehicle controls. N = 5 per group.</p

MN1 overexpression and p53 knockdown induce resistance in AML <i>in vitro</i>.

<p>A. Confirmation of KD in M1p5 cells. B. M1p5 shP53 or GFP cells were exposed to a titration of Didox (0–25 µM) for 72 hours and viability assays performed. C. MFL2 shP53 or GFP cells were exposed to a titration of Didox (0–40 µM) for 72 hours and viability assays performed. D. 3 independent 72 hour viability assays with MN1 and GFP controls, in triplicate with titrations of Didox (0–50 µM).*  = p of value less than 0.05.</p

Inhibitory concentrations of murine and human AML.

<p>Inhibitory concentrations of murine and human AML.</p

Didox has activity in AML models <i>in vivo</i>.

<p>A. Schema. 1.0×10⁶ luciferase tagged AML cells were injected into sublethally irradiated (4.5 Gy) recipients and allowed to engraft. Engraftment was monitored by bioluminescent imaging (IVIS 100 imager). Animals received 5 days of Didox at 425 mg/kg or D5 water control via intraperitoneal injection (IP). Animals were followed for survival. B. Representative bioluminescent images from Nras^G12D (MR2) mice pre- and post-treatment. C. Quantitation of bioluminescence post-treatment. D. Kaplan-Meier survival curves of Didox <i>in vivo</i> studies from start of treatment. *  = p of value less than 0.05.</p

Didox has activity against AML in colony formation assays.

<p>A. Didox reduced colony formation in KG1a cells. Cells were exposed to titration of Didox for 24 hours before incubation in methylcellulose (7–8 days). Mean colony formation was assessed in triplicate in 3 experiments and normalized to untreated controls. B. Didox exposure reduced colony formation in primary AML samples. Primary samples (C1–C3) were exposed to a titration of Didox for 24 hours before incubation in methylcellulose (12–14 days). Mean colony formation was assessed in triplicate in 3 experiments and normalized to untreated controls. *  = p value less than 0.05.</p

RR is expressed in AML.

<p>A. Western blots performed for RR small subunit. AML patient samples (bone marrow, M1–M3, leukopheresis A1–A5), and cell lines (K – KG1a, M – MFL2, H – HL-60, K5 – K562). B. Growth curves. Cell lines were treated with Didox for 72 hours. Viability was assessed and normalized to untreated controls.</p