肿瘤细胞治疗研究
英文原题：Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.
这些发现表明NX-1607对Cbl-b的抑制可能增强MAPK/ERK信号通路的激活，从而维持T细胞活化。
背景：E3泛素连接酶casitas B淋巴瘤-b（Cbl-b）通过减弱T细胞活化和细胞因子产生，在调节免疫反应中起关键作用。抑制Cbl-b通过增强免疫活性，在免疫肿瘤学中是一种潜在的治疗策略。方法：采用快速均相时间分辨荧光（HTRF）实验评估NX-1607对Cbl-b的抑制效果。使用原代T细胞和Jurkat T细胞在体外表征NX-1607对T细胞活化、细胞因子产生和增殖的影响。利用CD69标志物通过流式细胞术进行药物组合筛选，以确定参与NX-1607诱导T细胞活化的信号通路。使用CRISPR/Cas9技术在Jurkat T细胞中敲除PLCG1和MAPK3/1，随后通过Western blotting检测p-PLCγ1和p-ERK1/2。在BALB/c小鼠的A20 B细胞淋巴瘤模型中评估NX-1607的抗肿瘤疗效，随后进行流式细胞术分析以检查TIL（肿瘤浸润淋巴细胞）（TILs）的表型。结果：我们的数据显示，NX-1607在低纳摩尔水平有效抑制Cbl-b活性，增强PLCγ1和HCSL1磷酸化，激活MAPK/ERK信号传导，并提高CD69表达。抑制PLCγ1和ERK1/2显著降低NX-1607对T细胞活化的影响。口服给予NX-1607显著降低A20 B细胞淋巴瘤模型中的肿瘤生长，TIL（肿瘤浸润淋巴细胞）的免疫表型分析显示治疗肿瘤中CD3+、CD4+和CD8+ T细胞增加。此外，我们的结果表明，NX-1607治疗导致循环T细胞中磷酸化PLCγ1和ERK1/2水平升高。结论：综上所述，这些发现表明NX-1607对Cbl-b的抑制可能增强MAPK/ERK信号通路的激活，从而维持T细胞活化。这为NX-1607的分子机制提供了有力证据，强调了Cbl-b在T细胞受体（TCR）结合后控制T细胞活化信号强度中的关键作用。
Backgroud: The E3 ubiquitin ligase casitas B lymphoma-b (Cbl-b) is pivotal in modulating immune responses by attenuating T-cell activation and cytokine production. Inhibiting Cbl-b presents a potential therapeutic strategy in immuno-oncology by enhancing immune activity. Methods: A rapid Homogeneous Time-Resolved Fluorescence (HTRF) assay was employed to evaluate the inhibitory efficacy of NX-1607 on Cbl-b. The effects of NX-1607 on T cell activation, cytokine production, and proliferation were characterized invitro using primary T cells and Jurkat T cells . A drug combination screening was performed utilizing the CD69 marker via flow cytometry to dentify signaling pathways involved in T cell activation by NX-1607. CRISPR/Cas9 technology was used to knock out PLCG1 and MAPK3/1 in Jurkat T cells, followed by the detection of p-PLCγ1 and p-ERK1/2 though Western blotting. The antitumor efficacy of NX-1607 was assessed in a murine model of A20 B-cell lymphoma using BALB/c mice, with subsequent flow cytometry analysis conducted to examine the phenotype of tumor-infiltrating lymphocytes (TILs). Results: Our data show that NX-1607 effectively inhibits Cbl-b activity at low nanomolar levels, boosting PLCγ1 and HCSL1 phosphorylation, activating MAPK/ERK signaling, and elevating CD69 expression. Inhibiting PLCγ1 and ERK1/2 significantly reduces NX-1607's effect on T-cell activation. Oral administration of NX-1607 notably decreases tumor growth in the A20 B-cell lymphoma model, with immunophenotyping analyses of tumor-infiltrating lymphocytes revealing increased CD3 + , CD4 + , and CD8 + T cells in treated tumors. Furthermore, our results demonstrate that treatment with NX-1607 results in increased levels of phosphorylated PLCγ1 and ERK1/2 in circulating T cells. Conclusion: Taken together, these findings imply that the inhibition of Cbl-b by NX-1607 may enhance the activation of the MAPK/ERK signaling pathway, thereby sustaining T-cell activation. This provides compelling evidence for the molecular mechanism of NX-1607, underscoring the pivotal role of Cbl-b in controlling signal strength in T-cell activation after T-cell receptor (TCR) engagement.
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