弥漫性大 B 细胞淋巴瘤 (DLBCL) 是一种常见的恶性肿瘤，大约 40% 的患者因利妥昔单抗耐药而面临治疗挑战或复发，这主要是由于 CD20 表达减少或缺失。我们之前的研究发现 PDK4 通过对 CD20 表达的负调节而成为利妥昔单抗耐药性的关键驱动因素。对PDK4耐药机制的进一步研究和先进外泌体纳米颗粒复合物的开发可能会揭示新的耐药靶点，并为创新、有效的DLBCL治疗方式铺平道路。我们利用了具有高PDK4表达的DLBCL耐药细胞系（SU-DHL-2） /R）。我们用短发夹 RNA (shRNA) 慢病毒感染它进行 RNA 测序，旨在识别耐药细胞中显着下调的 mRNA。采用免疫荧光、免疫组织化学和蛋白质印迹等技术确定 PDK4 在耐药细胞中的定位和表达及其在组蛋白脱乙酰酶 8 (HDAC8) 磷酸化中的调节作用。此外，我们通过细胞、遗传和化学工程方法设计了先进的外泌体纳米颗粒复合物aCD20@ExoCTX/siPDK4。这些纳米颗粒通过动态光散射 (DLS) 和透射电子显微镜 (TEM) 进行表征，并通过流式细胞术评估它们的细胞摄取。我们使用 CCK-8 测定和流式细胞术评估了纳米颗粒对 DLBCL 耐药细胞和免疫细胞凋亡的影响。此外，还在耐药 DLBCL 小鼠模型中测试了它们抵抗耐药性和发挥抗肿瘤作用的能力。我们发现 PDK4 通过磷酸化 Ser-39 位点启动 HDAC8 激活，通过脱乙酰化抑制 CD20 蛋白表达。 aCD20@ExoCTX/siPDK4纳米粒子作为基因治疗和单克隆抗体的有效细胞内递送机制，同时诱导耐药DLBCL细胞凋亡并触发肿瘤细胞中的免疫原性细胞死亡。这种双重作用有效逆转了免疫抑制的肿瘤微环境，在皮下小鼠肿瘤耐药模型中显示出协同治疗效果。这项研究表明，PDK4 通过 HDAC8 磷酸化调节 CD20 表达，有助于 DLBCL 中的利妥昔单抗耐药。设计的外泌体纳米颗粒通过靶向 PDK4/HDAC8/CD20 途径有效克服了这种耐药性，代表了一种有前途的药物递送方法和治疗利妥昔单抗耐药性 DLBCL 患者。© 2024。作者。

Diffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor, with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance, primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4's resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative, effective treatment modalities for DLBCL.We utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing, aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence, immunohistochemistry, and Western blotting were employed to determine PDK4's localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore, we engineered advanced exosome nanoparticle complexes, aCD20@ExoCTX/siPDK4, through cellular, genetic, and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles' effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally, their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model.We found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site, suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies, simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment, showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model.This study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway, representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.© 2024. The Author(s).