Zereshkian Arman, Leyton Jeffrey V, Cai Zhongli, Bergstrom Dane, Weinfeld Michael, Reilly Raymond M
Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada; Department of Nuclear Medicine and Radiobiology, Université de Sherbrooke, Sherbrooke, QC, Canada.
Nucl Med Biol. 2014 May-Jun;41(5):377-83. doi: 10.1016/j.nucmedbio.2014.02.003. Epub 2014 Feb 14.
Leukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131⁻ phenotype uniquely displayed by LSCs.
The surviving fraction (SF) of CD123⁺/CD131⁻ AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33-266 nmols/L; 0.74MBq/μg) or to γ-radiation (0.25-5Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) or with γ-radiation (0.25-2Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation.
Binding of (111)In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131⁻ epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25-0.5Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6Gy to AML-5 cells.
We conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.
白血病干细胞（LSCs）被认为是引发和传播急性髓系白血病（AML）以及导致治疗后复发的原因。靶向这些细胞的放射免疫疗法（RIT）可能会改善AML的治疗，但受到靶抗原表位低密度的限制。我们的目标是研究一种干扰DNA修复的人多核苷酸激酶/磷酸酶（hPNKP）抑制剂，作为俄歇电子RIT药物¹¹¹In-NLS-7G3的放射增敏剂，该药物能特异性识别LSCs独特显示的CD123⁺/CD131⁻表型。
通过克隆形成试验测定暴露于¹¹¹In-NLS-7G3（33 - 266 nmol/L；0.74MBq/μg）或γ射线（0.25 - 5Gy）的CD123⁺/CD131⁻ AML-5细胞的存活分数（SF）。评估A12B4C3（25 μmol/L）与¹¹¹In-NLS-7G3（16 - 66 nmol/L）或γ射线（0.25 - 2Gy）联合使用对AML-5细胞SF的影响。使用γ-H2AX试验测量细胞核中DNA双链断裂（DSB）的密度。基于通过细胞分级分离测量的¹¹¹In-NLS-7G3的亚细胞分布估计细胞剂量学。
在存在过量（1μM）未标记的NLS-7G3的情况下，¹¹¹In-NLS-7G3与AML-5细胞的结合减少了2.2倍，表明其与CD123⁺/CD131⁻抗原表位特异性结合。¹¹¹In-NLS-7G3使AML-5细胞的SF从33 nmol/L时的86.1±11.0%降至266 nmol/L时的10.5±3.6%。未标记的NLS-7G3对SF无显著影响。用γ射线处理AML-5细胞使SF从0.25Gy时的98.9±14.9%降至5Gy时的0.03±0.1%。与单独使用放射免疫缀合物相比，A12B4C3与¹¹¹In-NLS-7G3（16 - 66 nmol/L）联合使用使细胞毒性增强高达1.7倍，并与细胞核中DNA DSB增加1.6倍相关。A12B4C3使γ射线（0.25 - 0.5Gy）对AML-5细胞的细胞毒性增强高达1.5倍，DNA DSB增加1.7倍。暴露于¹¹¹In-NLS-7G3（66 nmol/L）给AML-5细胞带来高达0.6Gy的剂量。
我们得出结论，A12B4C3使AML细胞对¹¹¹In-NLS-7G3的DNA损伤作用产生放射增敏作用。联合治疗可能会提高针对LSC亚群的AML的俄歇电子RIT的有效性。
