基于阻断IL-6介导的信号转导探讨托珠单抗增强微环境中AML细胞化疗敏感性的机制研究

The bone marrow microenvironment can protect acute myeloid leukemia (AML) cells from injury by therapeutic drugs, thus partly causing AML relapse. How to effectively restore the drug sensitivity in AML cells in microenvironment is still unclear. Our previous study showed that adhesion to stromal cells induced multidrug resistance in AML cells partially via activating IL-6 mediated signal transduction abnormally. Silencing IL-6 in HS-5 cells reversed the adhesion induced resistance in AML cells. Based on these results, we hypothesized Tocilizumab (TCZ), a humanized antihuman IL-6R antibody, can reverse the adhesion induced resistance via blocking IL-6 mediated signal pathway. To prove this hypothesis, the following will be studied :① in co-culture system added with TCZ, observe the effect on drug resistance in AML cells and the effect on cell proliferation, apoptosis and cell cycle in AML and stroma cells; ② the protein chip technology was used to screen the vital molecule of TCZ on AML cells adhered to stroma cells and further confirmed by RNA interference; ③ the effect of the combination of TCZ and cytarabine （Ara-C） in vivo through setting up a mouse AML model. The research would provide theory basis and feasible measure for the combination of TCZ and Ara-C.

微环境可为急性髓系白血病（AML）细胞提供庇护助其化疗逃逸，成为复发的重要根源。如何有效恢复微环境中AML细胞化疗敏感性目前尚不明确。我们前期发现粘附基质细胞生长的 AML细胞对多种化疗药物敏感性下降，与IL-6介导的信号通路在粘附共培养过程中异常激活密切相关，沉寂基质细胞IL-6可有效逆转共培养诱导的化疗抵抗。因此本课题将在此基础上深入研究IL-6受体单抗—托珠单抗（TCZ）增强微环境AML细胞化疗敏感性的机制，通过：①TCZ作用于粘附共培养体系，观察TCZ对AML细胞药物敏感性的改变及对AML细胞、基质细胞的影响；②运用蛋白芯片技术筛选TCZ作用于AML细胞后IL-6信号通路中的关键分子，并用RNA干扰技术进一步确认；③构建人AML小鼠模型，探讨TCZ与化疗药物联合的体内效应。本项目将阐明TCZ增强微环境中AML细胞化疗敏感性的分子机制，为TCZ与化疗方案联合应用提供理论依据。

课题组前期研究发现IL-6在骨髓微环境诱导的急性髓系白血病（AML）细胞的药物抵抗中扮演了重要角色，为此本研究探讨IL-6受体单抗-托珠单抗（TCZ）能否有效逆转逆转骨髓微环境所诱导的AML细胞药物抵抗，并探讨相关机制。本研究主要包括：1）观察初诊、复发、缓解AML患者及正常健康供者骨髓基质细胞上清中IL-6水平；2）构建IL-6基因敲低的HS-5细胞株，比较AML细胞粘附于野生型和IL-6敲除后基质细胞的药物敏感性差异；3）观察托珠单抗（TCZ）对基质细胞所诱导AML药物抵抗的逆转；4）Western Blot法检测共培养前后及托珠单抗作用后IL-6-JAK-STAT3信号通路的改变；5）构建人AML小鼠皮下荷瘤模型，体内探讨TCZ协助Ara-C杀伤白血病细胞的作用机制。结果显示：1）初诊、复发AML患者骨髓基质细胞培养上清中IL-6水平高于缓解AML患者和健康供者；2）与敲低IL-6的人骨髓基质细胞HS-5进行黏附共培养，可减少骨髓基质细胞诱导的AML细胞药物抵抗;3）粘附共培养可显著降低AML细胞株（U937、HL-60）对Ara-C、DNR和HHT的化疗敏感性，单独给药的TCZ对粘附共培养前后的AML细胞无明显抑制作用，TCZ协助作用后可逆转骨髓微环境所诱导药物抵抗；4） 粘附共培养后原癌基因信号传导及转录激活蛋白3(signal transducer and activator of transcription 3, STAT3)升高，且随着时间延长逐渐增加，TCZ可抑制粘附共培养后AML细胞内升高的STAT3，与其磷酸化蛋白p-STAT3，以及PI3K、ERK信号通路中的重要磷酸化蛋白p-AKT、p-ERK 1/2；5）小鼠体内研究表明，TCZ可协同Ara-C清除体内HL-60白血病细胞，并显著抑制荷瘤小鼠瘤体体积的增长；通过免疫组化法检测到，IL-6-JAK-STAT3轴关键蛋白分子STAT3、p-STAT3、p-AKT以及p-ERK 1/2在TCZ作用下表达减弱。本研究通过体外体内研究，发现IL-6-JAK-STAT3轴异常激活参与骨髓基质细胞所诱导AML细胞耐药，TCZ可通过抑制该轴的激活，增强AML细胞的化疗敏感性。本研究将为托珠单抗运用于临床AML的治疗提供理论依据。