Department of Spine Surgery, Third Xiangya Hospital, Central South University, Changsha, Hunan, China.
Department of Hematology, Liuzhou People's Hospital affiliated to Guangxi Medical University, Liuzhou, Guangxi, China.
Mol Cancer. 2024 Jul 15;23(1):144. doi: 10.1186/s12943-024-02057-0.
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@Exo/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@Exo/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.
弥漫性大 B 细胞淋巴瘤（DLBCL）是一种常见的恶性肿瘤，约有 40%的患者因利妥昔单抗耐药而面临治疗挑战或复发，主要原因是 CD20 表达减少或缺失。我们之前的研究发现 PDK4 通过负调控 CD20 的表达，成为利妥昔单抗耐药的关键驱动因素。进一步研究 PDK4 的耐药机制和先进的外泌体纳米颗粒复合物的开发可能揭示新的耐药靶点，并为 DLBCL 开辟创新、有效的治疗途径。
我们利用高 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，旨在鉴定耐药细胞中显著下调的 mRNA。我们采用免疫荧光、免疫组织化学和 Western blot 等技术，确定 PDK4 在耐药细胞中的定位和表达，及其在组蛋白去乙酰化酶 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@Exo/siPDK4。通过动态光散射（DLS）和透射电子显微镜（TEM）对这些纳米颗粒进行了表征，并通过流式细胞术评估了它们在细胞内的摄取情况。我们使用 CCK-8 测定法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，我们还在耐药 DLBCL 小鼠模型中测试了它们逆转耐药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 的激活，通过去乙酰化抑制 CD20 蛋白表达。aCD20@Exo/siPDK4 纳米颗粒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，并引发肿瘤细胞免疫原性细胞死亡。这种双重作用有效地逆转了免疫抑制性肿瘤微环境，在皮下小鼠肿瘤耐药模型中表现出协同治疗效果。
本研究表明，PDK4 通过磷酸化 HDAC8 调节 CD20 表达，导致 DLBCL 对利妥昔单抗耐药。设计的外泌体纳米颗粒通过靶向 PDK4/HDAC8/CD20 通路有效克服这种耐药性，为药物输送和治疗利妥昔单抗耐药的 DLBCL 患者提供了一种有前途的方法。
