目的：探讨β-catenin/CREB结合蛋白（CBP）通路抑制剂PRI-724联合伊马替尼对BCR-ABL1阳性慢性髓系白血病（CML）细胞及荷瘤模型的抗肿瘤作用及其潜在机制。方法：体外实验采用人K562细胞、能够稳定表达野生型BCR-ABL1（32D-WT）和T315I突变型BCR-ABL1（32D-T315I）的鼠源32D髓系细胞。采用CCK-8法检测伊马替尼、PRI-724单药及联合处理对细胞存活能力的影响，并计算半数抑制浓度（IC50）和联合指数（CI）；采用SA-β-gal染色检测细胞衰老相关表型；采用免疫共沉淀（Co-IP）法检测PRI-724对β-catenin与CBP/p300相互作用的影响。体内实验采用K562、32D-WT和32D-T315I细胞建立裸鼠皮下荷瘤模型，分别设未治疗组、伊马替尼组、PRI-724组及伊马替尼+PRI-724组，并给予相应处理，观察瘤体生长变化，并基于肿瘤生长抑制率（TGI）计算体内CI。采用WB法检测瘤组织中PI3K/AKT信号通路相关蛋白、核内β-catenin/CBP及下游效应因子c-Myc、Cyclin D1的表达。结果：伊马替尼和PRI-724对K562、32D-WT和32D-T315I细胞均呈浓度依赖性抑制作用。伊马替尼对K562、32D-WT和32D-T315I细胞的IC50分别为0.860、0.594和1.646 μmol/L；PRI-724对上述3种细胞的IC50分别为1.120、0.761和0.756 μmol/L。与32D-WT细胞相比，32D-T315I细胞对伊马替尼的敏感性降低，而对PRI-724的敏感性未见明显下降。K562、32D-WT和32D-T315I细胞中伊马替尼 + PRI-724联合处理的CI分别为0.635、0.780和0.689，提示两药在体外具有协同抑制作用。SA-β-gal染色显示，与伊马替尼单药组相比，PRI-724单药组3种CML细胞β-gal阳性细胞比例均升高，联合伊马替尼后进一步升高。Co-IP结果显示，PRI-724处理后β-catenin免疫沉淀复合物中CBP共沉淀水平降低，而p300共沉淀水平升高。体内实验显示，与伊马替尼单药组相比，伊马替尼 + PRI-724联合组瘤体体积和瘤体质量进一步降低；但基于TGI计算的K562、32D-WT和32D-T315I荷瘤模型体内CI均 > 1，提示在当前剂量和给药方案下，两药体内联合效应未达到理论相加预期，可能存在拮抗趋势。WB结果显示，与伊马替尼单药组相比，联合治疗组瘤体组织中p-PI3K/PI3K、p-AKT/AKT比值降低，核内β-catenin、CBP、c-Myc和Cyclin D1表达水平均降低。结论：PRI-724联合伊马替尼在体外具有协同抑制作用，并可在体内较伊马替尼单药进一步抑制瘤体生长。其机制可能与调节β-catenin/CBP/p300复合物、抑制PI3K/AKT信号通路及下调c-Myc、Cyclin D1表达有关。

Objective: To investigate the antitumor effects and potential mechanisms of the β-catenin/CREB-binding protein (CBP) pathway inhibitor PRI-724 combined with imatinib in BCR-ABL1-positive chronic myeloid leukemia (CML) cells and xenograft models. Methods: Human K562 cells and murine 32D myeloid cells stably expressing wild-type BCR-ABL1 (32D-WT) or T315I-mutant BCR-ABL1 (32D-T315I) were used for in vitro experiments. The effects of imatinib, PRI-724, and their combination on cell viability were detected using the CCK-8 assay, and the half-maximal inhibitory concentration (IC50) and combination index (CI) were calculated. Senescence-associated phenotypes were examined by SA-β-gal staining. Co-immunoprecipitation (Co-IP) was used to detect the effect of PRI-724 on the interaction between β-catenin and CBP/p300. For in vivo experiments, subcutaneous xenograft models were established in nude mice using K562, 32D-WT, and 32D-T315I cells. Untreated, imatinib, PRI?724, and imatinib + PRI?724 groups were established, and each group received the corresponding treatment, and tumor growth was monitored. The in vivo CI was calculated based on tumor growth inhibition (TGI). WB was used to detect the expression of PI3K/AKT signaling pathway-related proteins, nuclear β-catenin/CBP, and downstream effectors c-Myc and Cyclin D1 in tumor tissues. Results: Imatinib and PRI-724 inhibited K562, 32D-WT, and 32D-T315I cells in a concentration-dependent manner. The IC50 values of imatinib in K562, 32D-WT, and 32D- T315I cells were 0.860, 0.594, and 1.646 μmol/L, respectively; the IC50 values of PRI-724 in the above three cell types were 1.120, 0.761, and 0.756 μmol/L, respectively. Compared with 32D-WT cells, 32D-T315I cells showed reduced sensitivity to imatinib, whereas no obvious decrease in sensitivity to PRI-724 was observed. The CI values of imatinib + PRI-724 in K562, 32D-WT, and 32D-T315I cells were 0.635, 0.780, and 0.689, respectively, suggesting a synergistic inhibitory effect in vitro. SA- β-gal staining showed that, compared with imatinib monotherapy, PRI-724 monotherapy increased the proportion of SA- β-gal-positive cells in all three cell models, and the combination further increased this proportion. Co-IP results showed that, after PRI-724 treatment, the level of CBP co-precipitated with β-catenin was decreased, whereas the level of p300 co-precipitated with β-catenin was increased. In vivo experiments showed that, compared with the imatinib monotherapy group, the imatinib + PRI-724 combination group showed further reductions in tumor volume and tumor weight. However, the in vivo CI values calculated using the TGI-based response-additivity approach in the K562, 32D-WT, and 32D-T315I xenograft models were all > 1, suggesting that, under the current dose and administration regimen, the in vivo combination effect of the two drugs did not reach the expected additive effect and may have shown an antagonistic trend. WB showed that, compared with the imatinib monotherapy group, the combination treatment group had lower p-PI3K/PI3K and p-AKT/ AKT ratios and lower expression levels of nuclear β-catenin, CBP, c-Myc, and Cyclin D1 in tumor tissues. Conclusion: PRI-724 combined with imatinib exhibits a synergistic inhibitory effect in vitro and further suppresses tumor growth in vivo compared with imatinib monotherapy. The underlying mechanism may be related to modulation of the β-catenin/CBP/p300 complex, inhibition of the PI3K/AKT signaling pathway, and downregulation of c-Myc and Cyclin D1 expression.

银川市科技计划（2024SF014）；宁夏自然科学基金（2024AAC03782）