由于针对复发和难治性B细胞急性淋巴细胞白血病（B-ALL）的有效治疗方案仍然存在问题，因此需要新的治疗策略。 Artemis 是 V(D)J 重组和非同源末端连接（NHEJ）DNA 双链断裂（DSB）修复中的关键内切酶。抑制 Artemis 在RAG表达的T和B细胞成熟期间会导致染色体断裂。尽管这会暂时阻止新的B和T细胞的产生，但它可以通过引起这些RAG表达的肿瘤细胞的染色体断裂从而在白血病和T-ALL细胞的增殖中具有肿瘤学益处。目前，对于 Artemis 还没有药理学上的抑制方法。根据对207名高危预B急性淋巴细胞白血病患儿的基因表达分析，高表达的 Artemis 与预后不良相关。因此，我们评估了四种化合物（827171、827032、826941和825226），这些化合物是从大规模的 Artemis 靶向药物筛选中生成的。使用纯化的 Artemis：DNA-PKcs 复合物的生化检测表明，Artemis 抑制剂 827171、827032、826941、825226 对阻断 Artemis 具有纳摩尔 IC50 值。我们将这四种化合物与 DNA-PK 抑制剂（AZD7648）在三种来自患者的B-ALL细胞株（LAX56、BLQ5和LAX7R）和两种成熟B细胞株（3301015和5680001）进行了比较。我们发现，药理学上的 Artemis 抑制显著降低了B-ALL细胞株的增殖，而正常的成熟B细胞株没有明显受到影响。使用DNA-PKcs（调节 Artemis）抑制剂 AZD7648 对这些主要的来自患者的所有细胞系的影响较小，表明需要直接抑制 Artemis，而不是间接抑制调节 Artemis 的激酶，才能实现 V(D)J 头发夹开放的抑制。我们的数据为进一步评估药理学抑制 Artemis 对 B-ALL 和 T-ALL 的增殖提供了基础。 版权所有 © 2023 Ogana、Hurwitz、Hsieh、Geng、Müschen、Bhojwani、Wolf、Larocque、Lieber 和 Kim。

As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds (827171, 827032, 826941, and 825226), previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines (LAX56, BLQ5 and LAX7R) and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL.Copyright © 2023 Ogana, Hurwitz, Hsieh, Geng, Müschen, Bhojwani, Wolf, Larocque, Lieber and Kim.