Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.
Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.
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转化的JAK2V617F激酶经常与骨髓增殖性肿瘤（mpn）相关，并被认为是髓系细胞过度产生的工具。一些靶向JAK2的小分子药物目前正处于临床开发阶段，用于治疗这些疾病。我们进行了高通量体外筛选，以确定JAK2V617F中可能具有潜在临床相关性并与JAK2抑制剂ruxolitinib （INCB018424）耐药相关的点突变。使用基于BaF3细胞的测定方法，筛选了七个突变JAK2V617F cDNA文库，以特异性鉴定预测药物结合区域的突变，这些突变将赋予对鲁索利替尼的耐药性。我们确定了5种不同的非同义点突变，赋予耐药性。含有突变的细胞对ruxolitinib的EC50比原生JAK2V617F高9至33倍。我们的研究结果进一步表明，这些突变还赋予了对所有JAK2激酶抑制剂的交叉耐药，包括AZD1480、TG101348、莱司替尼（cap -701）和CYT-387。令人惊讶的是，在JAK2V617F中引入“守门人”突变（M929I）仅影响鲁索利替尼的敏感性（EC50增加4倍）。这些结果表明，目前处于临床试验中的JAK2抑制剂可能由于点突变而容易产生耐药性，在使用这些药物时应谨慎。
The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified 5 different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib compared to native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the ‘gatekeeper’ mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.
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