FLT3-ITD tyrosine kinase inhibitors (TKI) show limited clinical activity in acute myeloid leukemia (AML) due to emerging resistance. TKI resistance is mediated by secondary FLT3-ITD mutations only in a minority of cases. We hypothesize that the cytokine CCL5 protects AML cells from TKI-mediated cell death and contributes to treatment resistance. We generated PKC412- and sorafenib-resistant MOLM-13 cell lines as an in vitro model to study TKI resistance in AML. Increased CCL5 levels were detected in supernatants from PKC412-resistant cell lines compared to TKI-sensitive cells. Moreover, CCL5 treatment of TKI-sensitive cells induced resistance to PKC412. In resistant cell lines with high CCL5 release, we observed a significant downregulation of the CCL5-receptor CCR5 and CXCR4. In these cell lines, TKI resistance could be partly overcome by addition of the CXCR4-receptor antagonist plerixafor. Microarray and intracellular flow cytometry analyses revealed increased p-Akt or p-Stat5 levels in PKC412-resistant cell lines releasing high amounts of CCL5. Treatment with the CXCR4 antagonist plerixafor, αCCL5, or CCR5-targeting siRNA led to a decrease of p-Akt-positive cells. Transient transfection of sensitive MOLM-13 cells with a CCL5-encoding vector mediated resistance against PKC412 and led to an increase in p-Akt-positive and p-Stat5-positive cells. Isolated AML blasts from patients treated with PKC412 revealed that CCL5 transcript levels increase significantly at relapse. Taken together, our findings indicate that CCL5 mediates resistance to FLT3-TKIs in FLT3-ITD-mutated AML and could possibly serve as a biomarker to predict drug resistance.

中文翻译：

---

CCL5介导FLT3-ITD阳性AML中对FLT3抑制剂的靶激酶依赖性耐药。

由于出现耐药性，FLT3-ITD酪氨酸激酶抑制剂（TKI）在急性髓细胞性白血病（AML）中显示出有限的临床活性。仅在少数情况下，TKI耐药性由继发性FLT3-ITD突变介导。我们假设细胞因子CCL5保护AML细胞免受TKI介导的细胞死亡，并有助于治疗抗性。我们生成了PKC412耐药和索拉非尼耐药的MOLM-13细胞系作为体外模型，以研究AML中的TKI耐药性。与TKI敏感细胞相比，在PKC412耐药细胞系的上清液中检测到CCL5水平升高。此外，TCL敏感细胞的CCL5处理诱导了对PKC412的抗性。在具有高CCL5释放的抗性细胞系中，我们观察到CCL5受体CCR5和CXCR4的显着下调。在这些细胞系中 通过添加CXCR4受体拮抗剂plerixafor，可以部分克服TKI耐药性。微阵列和细胞内流式细胞仪分析显示，PKC412耐药细胞系中p-Akt或p-Stat5水平升高，释放出大量的CCL5。用CXCR4拮抗剂plerixafor，αCCL5或靶向CCR5的siRNA处理可导致p-Akt阳性细胞减少。用编码CCL5的载体瞬时转染敏感的MOLM-13细胞介导了对PKC412的抗性，并导致p-Akt阳性和p-Stat5阳性细胞增加。从用PKC412治疗的患者中分离出的AML母细胞显示，CCL5转录水平在复发时显着增加。综上所述，我们的发现表明CCL5介导了FLT3-ITD突变AML中对FLT3-TKIs的耐药性，并且可能充当预测耐药性的生物标记。