Table 4_Insights into the olaparib-mediated cell death mechanisms in canine hematological malignancies: a different fate for CLBL-1 and GL-1 cell lines.xlsx

IntroductionOlaparib (OLA) is a poly ADP-ribose polymerase inhibitor (PARPi) indicated for solid cancers harboring BRCA1/2 mutations. Recent evidences suggest that sensitivity to PARPis may also be influenced by other factors that impair the DNA repair mechanisms. Since various hematological malignancies exhibit these types of defects, this study aims to investigate further the mechanism of action of OLA in CLBL-1 and GL-1 canine cell lines, which showed different sensitivities to this PARPi.
MethodsCLBL-1 and GL-1 cell lines were exposed to OLA (12.5, 25, and 50 μM) for 24 and 48 h and were subjected to preliminary cell death evaluations by flow cytometry. Then, both immunoblotting for the assessment of Bcl-2 and Bcl-XL, and RNA-seq were carried out after 24 h of exposure to OLA 25 and 50 μM. As for whole-transcriptome analysis, reads were pseudo-aligned (Kallisto) to the reference transcriptome, and differential gene expression (DGE) and functional analyses were performed with edgeR and clusterProfiler R packages.
ResultsThe percentage of annexin V-positive cells after 24 h of incubation with OLA 50 μM was ~10%, increasing to ~40% in CLBL-1 cells and ~30% in GL-1 cells at 48 h. Bcl-2 and Bcl-XL expression increased after 24 h of incubation in CLBL-1 cells but decreased in GL-1 cells. DGE and functional analyses showed that, in CLBL-1 cells, the main processes affected by OLA were stress (e.g., ATF3, CEBPB) and apoptosis (e.g., BAX, BBC3). Conversely, in GL-1 cells, the regulation of tumor necrosis factor and interferon response-related terms, along with the upregulation of genes such as IL6, TNF, IFIT3, GSDME, and IL18, indicated the induction of pyroptosis.
DiscussionThe comprehensive transcriptomic analysis helped clarify the distinct mechanisms of OLA-induced cell death in CLBL-1 and GL-1 cells, which showed different sensitivities to OLA. Indeed, this PARPi appeared to interact with immune checkpoints, stress sensors, and interfere with cell proliferation, leading to various types of cell death. As canine lymphoma is a significant concern in veterinary oncology and a valuable model for its human counterpart, this study further confirms the potential of PARPi as a therapeutic approach in hematological malignancies in both species.

引言
奥拉帕利（Olaparib，OLA）属于聚ADP核糖聚合酶抑制剂（poly ADP-ribose polymerase inhibitor，PARPi），其获批适应症为携带BRCA1/2突变的实体恶性肿瘤。近期研究证据表明，PARPi的敏感性亦可受其他损伤DNA修复机制的因素影响。鉴于多种血液系统恶性肿瘤均存在此类DNA修复缺陷，本研究旨在进一步探究奥拉帕利在对该PARPi呈现不同敏感性的犬类细胞系CLBL-1与GL-1中的作用机制。

方法
将CLBL-1与GL-1细胞系分别置于浓度为12.5、25及50 μM的奥拉帕利环境中培养24 h与48 h，并通过流式细胞术初步评估细胞死亡情况。随后，在以25 μM与50 μM奥拉帕利处理24 h后，分别采用免疫印迹法检测Bcl-2与Bcl-XL的表达，并进行RNA测序（RNA-seq）分析。在全转录组分析环节，将测序reads通过Kallisto软件伪比对至参考转录组，并借助edgeR与clusterProfiler两款R包完成差异基因表达（differential gene expression, DGE）分析与功能富集分析。

结果
经50 μM奥拉帕利培养24 h后，膜联蛋白V（annexin V）阳性细胞占比约为10%；至48 h时，CLBL-1细胞与GL-1细胞的该阳性占比分别升至约40%与30%。CLBL-1细胞在奥拉帕利处理24 h后，Bcl-2与Bcl-XL的表达水平上调，而GL-1细胞中二者表达则出现下调。差异基因表达与功能分析结果显示，奥拉帕利对CLBL-1细胞的主要影响通路为应激反应（如ATF3、CEBPB）与细胞凋亡（如BAX、BBC3）。与之相反，GL-1细胞中肿瘤坏死因子与干扰素应答相关通路的调控，以及IL6、TNF、IFIT3、GSDME、IL18等基因的上调，提示奥拉帕利诱导了细胞焦亡。

讨论
本次全面的转录组分析阐明了奥拉帕利在对其呈现不同敏感性的CLBL-1与GL-1细胞中，诱导细胞死亡的差异化机制。该PARPi可与免疫检查点、应激感受器相互作用，并干扰细胞增殖，最终引发多种形式的细胞死亡。鉴于犬淋巴瘤在兽医肿瘤学中备受关注，且其可作为人类淋巴瘤的理想动物模型，本研究进一步证实了PARPi在两类物种的血液系统恶性肿瘤中作为治疗手段的应用潜力。