Macleod Alison C, Klug Lillian R, Patterson Janice, Griffith Diana J, Beadling Carol, Town Ajia, Heinrich Michael C
Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
Mol Cancer Ther. 2014 Dec;13(12):2840-51. doi: 10.1158/1535-7163.MCT-13-0830. Epub 2014 Sep 24.
Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.
耐药性KIT突变阻碍了KIT激酶抑制剂用于治疗系统性肥大细胞增多症患者的研发进程。本研究的目的是利用一组KIT突变的肥大细胞系，来表征一种新型联合疗法的协同效应，该联合疗法涉及抑制KIT和钙调神经磷酸酶（一种活化T细胞核因子（NFAT）调节剂）。评估了单一疗法或联合疗法对KIT突变肥大细胞的细胞活力/存活的影响。此外，在一个代表性细胞系中监测了NFAT依赖性转录活性，以评估联合疗法疗效的作用机制。最后，使用短发夹RNA（shRNA）稳定敲低钙调神经磷酸酶的表达，以确认钙调神经磷酸酶在观察到的协同作用中的作用。KIT抑制剂和钙调神经磷酸酶抑制剂（CNPI）联合使用可协同降低六种不同KIT突变肥大细胞系的细胞活力并诱导细胞凋亡。发现KIT抑制剂和CNPI均可降低NFAT依赖性转录活性。当与KIT抑制剂联合使用时，NFAT特异性抑制剂诱导的细胞凋亡与CNPI相似。值得注意的是，在每个测试的KIT突变细胞系中NFAT均呈组成性激活状态。敲低钙调神经磷酸酶亚基PPP3R1可使细胞对KIT抑制敏感，并增加NFAT磷酸化和细胞质定位。NFAT的组成性激活似乎代表了KIT突变肥大细胞疾病的一种新的可靶向特征。我们的研究表明，将KIT抑制与NFAT抑制相结合可能代表肥大细胞疾病的一种新治疗策略。
