Sheffield Cancer Research Centre, University of Sheffield, Sheffield, UK.
Bateson Centre, Department of Biomedical Science, University of Sheffield, Sheffield, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S98. doi: 10.1016/S0140-6736(15)60413-5.
The myeloproliferative neoplasms are a group of haematological malignancies characterised by pathological activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) intracellular signalling pathway. 50-95% of patients have an acquired mutation (JAK2V617F) causing constitutive activation of JAK2. Our aim was to find new treatments for myeloproliferative neoplasms by identifying compounds that suppress JAK/STAT pathway activation.
We used a luciferase-based transcriptional assay in the low complexity Drosophila model system to screen a library of 2000 small molecules for modulators of JAK/STAT pathway activation. Screen hits were validated with western blotting in the HDLM-2 Hodgkin's lymphoma cell line. The HEL cell line, in which constitutive JAK/STAT pathway activation is caused by JAK2V617F, was used to determine the relevance of screen hits for treatment of myeloproliferative neoplasms.
Methotrexate and the chemically similar drug aminopterin were independently identified as strong inhibitors of the Drosophila JAK/STAT pathway, an effect conserved to human cells. Methotrexate did not affect protein phosphorylation in other intracellular signalling pathways. Methotrexate caused significant suppression of JAK/STAT activation in HEL cells at a concentration equivalent to that seen in patients taking low-dose oral methotrexate (p≤0·001).
Our results suggest that methotrexate is a promising treatment for myeloproliferative neoplasms that could be translated into clinical trials after assessment in primary cells. These results are particularly relevant in myelofibrosis. Inhibitors of JAK1/2 improve symptoms and prolong life in myelofibrosis, but their use is limited by cost. Other existing therapies for myelofibrosis appear no more effective than placebo. Methotrexate might bring the benefits of JAK/STAT pathway inhibition at a lower cost.
Cancer Research UK, Yorkshire Cancer Research, UK Medical Research Council, Wellcome Trust, EU Framework Cancer Pathways.
骨髓增殖性肿瘤是一组血液系统恶性肿瘤，其特征在于 JAK/STAT（Janus 激酶和信号转导与转录激活因子）细胞内信号通路的病理性激活。50-95%的患者存在获得性突变（JAK2V617F），导致 JAK2 的组成性激活。我们的目的是通过鉴定抑制 JAK/STAT 通路激活的化合物，为骨髓增殖性肿瘤寻找新的治疗方法。
我们使用基于荧光素酶的转录测定法在低复杂度的果蝇模型系统中筛选了 2000 种小分子文库，以寻找 JAK/STAT 通路激活的调节剂。用 Western blot 在 HDLM-2 霍奇金淋巴瘤细胞系中验证筛选出的阳性化合物。用 HEL 细胞系（该细胞系中 JAK2V617F 导致组成性 JAK/STAT 通路激活）来确定筛选出的阳性化合物在治疗骨髓增殖性肿瘤方面的相关性。
甲氨蝶呤和结构相似的药物氨基喋呤被独立鉴定为果蝇 JAK/STAT 通路的强抑制剂，这种作用在人类细胞中也得到了保留。甲氨蝶呤不影响其他细胞内信号通路的蛋白磷酸化。甲氨蝶呤在相当于低剂量口服甲氨蝶呤患者（p≤0·001）的浓度下，显著抑制 HEL 细胞中 JAK/STAT 的激活。
我们的结果表明，甲氨蝶呤是一种有前途的骨髓增殖性肿瘤治疗药物，在经过原代细胞评估后，可以转化为临床试验。这些结果在骨髓纤维化中尤其相关。JAK1/2 抑制剂可改善骨髓纤维化的症状并延长患者生命，但由于成本限制，其应用受限。其他现有的骨髓纤维化治疗方法似乎并不比安慰剂更有效。甲氨蝶呤可能以较低的成本带来 JAK/STAT 通路抑制的益处。
英国癌症研究中心、约克郡癌症研究中心、英国医学研究理事会、威康信托、欧盟癌症途径框架。
