Department of Hematology, Jiangxi Provincial People's Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang 330006, China.
Medical College of Nanchang University, Nanchang 330006, China.
Leuk Res. 2024 Sep;144:107548. doi: 10.1016/j.leukres.2024.107548. Epub 2024 Jul 7.
Pirtobrutinib, a non-covalent Bruton's tyrosine kinase (BTK) inhibitor, has been approved as the first agent to overcome resistance to covalent BTK inhibitors (such as ibrutinib, acalabrutinib, and zanubrutinib). However, the mechanisms of pirtobrutinib resistance in chronic lymphocytic leukemia (CLL) remain poorly understood.
To investigate pirtobrutinib resistance, we established resistant cell models using BTK knock-out via CRISPR-Cas9 or chronic exposure to pirtobrutinib in MEC-1 cells. These models mimicked intrinsic or acquired resistance, respectively. We then analyzed differential protein expression between wild-type (WT) and resistant MEC-1 cells using Revers Phase Protein microArray (RPPA) and confirmed the findings through Western Blot. Additionally, we evaluated potential drugs to overcome pirtobrutinib resistance by conducting cell proliferation assays, apoptosis studies, and animal experiments using both sensitive and resistant cells.
MEC-1 cells developed resistance to pirtobrutinib either through BTK knock-out or prolonged drug exposure over three months. RPPA analysis revealed significant activation of proteins related to the PI3K/AKT pathway, including AKT and S6, in the resistant cells. Western Blot confirmed increased phosphorylation of AKT and S6 in pirtobrutinib-resistant MEC-1 cells. Notably, both the PI3K inhibitor (CAL101) and the AKT inhibitor (MK2206) effectively reduced cell proliferation and induced apoptosis in the resistant cells. The anti-tumor efficacy of these drugs was mediated by inhibiting the PI3K/AKT pathway. In vivo animal studies further supported the potential of targeting PI3K/AKT to overcome both intrinsic and acquired resistance to pirtobrutinib.
The PI3K/AKT pathway plays a crucial role in both intrinsic and acquired resistance to pirtobrutinib in CLL. Therapeutically targeting this pathway may offer a promising strategy to overcome pirtobrutinib resistance.
泊鲁替尼是一种非共价布鲁顿酪氨酸激酶（BTK）抑制剂，已被批准为克服共价 BTK 抑制剂（如伊布替尼、阿卡替尼和泽布替尼）耐药的第一种药物。然而，慢性淋巴细胞白血病（CLL）中泊鲁替尼耐药的机制仍知之甚少。
为了研究泊鲁替尼耐药性，我们通过 CRISPR-Cas9 敲除或在 MEC-1 细胞中慢性暴露于泊鲁替尼建立了 BTK 敲除的耐药细胞模型。这些模型分别模拟了内在或获得性耐药。然后，我们使用反相蛋白微阵列（RPPA）分析了 WT 和耐药 MEC-1 细胞之间的差异蛋白表达，并通过 Western Blot 验证了这些发现。此外，我们通过细胞增殖试验、凋亡研究以及使用敏感和耐药细胞进行的动物实验，评估了克服泊鲁替尼耐药的潜在药物。
MEC-1 细胞通过 BTK 敲除或长达三个月的药物暴露发展出对泊鲁替尼的耐药性。RPPA 分析显示，耐药细胞中与 PI3K/AKT 通路相关的蛋白，包括 AKT 和 S6，显著激活。Western Blot 证实了泊鲁替尼耐药的 MEC-1 细胞中 AKT 和 S6 的磷酸化增加。值得注意的是，PI3K 抑制剂（CAL101）和 AKT 抑制剂（MK2206）均能有效抑制耐药细胞的增殖并诱导凋亡。这些药物的抗肿瘤疗效是通过抑制 PI3K/AKT 通路介导的。体内动物研究进一步支持了靶向 PI3K/AKT 克服泊鲁替尼内在和获得性耐药的潜力。
PI3K/AKT 通路在 CLL 中对泊鲁替尼的内在和获得性耐药都起着至关重要的作用。靶向该通路可能是克服泊鲁替尼耐药的一种有前途的策略。
