The in vivo toxicity of hydroxyurea depends on its direct target catalase

Abstract

Hydroxyurea (HU) is a well tolerated ribonucleotide reductase inhibitor effective in HIV, sickle cell disease, and blood cancer therapy. Despite a positive initial response, however, most treated cancers eventually progress due to development of HU resistance. Although RNR properties influence HU resistance in cell lines, the mechanisms underlying cancer HU resistance in vivo remain unclear. To address this issue, we screened for HU resistance in the plant Arabidopsis thaliana and identified seventeen unique catalase mutants, thereby establishing that HU toxicity depends on catalase in vivo. We further demonstrated that catalase is a direct HU target by showing that HU acts as a competitive inhibitor of catalase-mediated hydrogen peroxide decomposition. Considering also that catalase can accelerate HU decomposition in vitro and that co-treatment with another catalase inhibitor alleviates HU effects in vivo, our findings suggests that HU could act as a catalase-activated pro-drug. Clinically, we found high catalase activity in circulating cells from untreated chronic myeloid leukemia, offering a possible explanation for the efficacy of HU against this malignancy.

FIGURE 1.

HU uptake by wild-type and HU-resistant mutants. 11-day-old wild-type and mutant seedlings grown on drug-free medium were moved to plates containing 25 mm HU and incubated for 8 or 24 h before shoots were harvested. After 24 h, the HU concentration in seedling shoots approached that of the medium for both wild-type and HU-resistant plants. HU could not be detected in plants grown on drug-free medium. Error bars indicate ±S.E.

FIGURE 2.

Inhibition of RNR activity by HU in wild-type and HU-resistant mutants. RNR activity in extracts from wild-type and HU-resistant mutants is shown. In the control, no HU was added to the RNR reaction. Where indicated, 1 or 5 mm HU was added to the RNR reaction. Error bars indicate ±S.E.

FIGURE 3.

HU response in wild-type and HU-resistant mutants. A, >90% of wild-type plants germinated on 2.5 mm HU, whereas <1/20,000 germinated on 3 mm HU. Two HU-resistant mutants germinating at significantly higher concentrations of HU than the wild type are shown. B and C, 7-day-old seedlings grown on 3 mm HU. B, wild type; C, cat2-9.

FIGURE 4.

HU inhibition of catalase-mediated decomposition of H₂O₂. A, catalase activity in plant and rat protein preparations in the absence (− HU) and presence of 1 mm HU (+ HU) is shown. Triplicate measurements were performed, and error bars indicate ±S.D. B, fractional activity, with respect to catalase activity under the same conditions in the absence of HU, is plotted versus log[c_HU (mm)]. The black line indicates the best fit to a sigmoidal response curve. C, catalase activity plotted versus c_H2O2 in the presence of varying concentrations of HU (millimolar). Black lines indicate the best fit to a competitive inhibition model.

FIGURE 5.

Co-treatment of A. thaliana seedlings with HU and 3-AT. A. thaliana seedlings germinated on medium containing 3 mm HU (A) and 3 mm HU and 20 μm 3-AT (B). Note that cotelydons are unfolded in B but not in A.

FIGURE 6.

Catalase activity in patient blood samples. Catalase activity in blood samples from a control group (Normal), patients diagnosed with chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelofibrosis (MF), myelomatosis (MM), unspecified myeloproliferative disease (MP), polycytaemia vera (PV), thrombocytosis (TC), chronic lymphatic leukemia (CLL), or lymphoma (L). Values were normalized to the average activity of the Normal group. Closed triangles indicate that patients were undergoing HU treatment at the time of sampling. Standard errors for technical replicates and more detailed diagnoses, where available, are shown in supplemental Table S1.