RON kinase as a novel therapeutic target in myeloproliferative neoplasms

摘要 费城染色体阴性的骨髓增生性肿瘤(MPN)，包括真性红细胞增多症 (PV)、原发性血小板增多症(ET)和原发性骨髓纤维化(PMF)是克隆性造血干细胞。 一种以一个或多个髓系骨髓室的增殖为特征的疾病。驱动因素突变 即激活Janus Kinase 2(JAK2)、血小板生成素受体(MPL)或 钙网蛋白(CALR)，功能是上调JAK/STAT信号。在疾病发展过程中，MPN患者 经历促炎细胞因子分泌增加，导致骨髓重塑 微环境和随后的纤维化。目前，治疗MPNS的唯一有效方法是干细胞。 移植，但大多数患者由于年龄或合并疾病而不是很适合移植。JAK抑制剂鲁索利替尼 是一种被批准的针对MPN患者的靶向疗法，并在减少脾肿大方面显示出希望。 以及在患者体内观察到的细胞因子风暴。然而，JAK抑制剂本身并不足以减少骨骼。 骨髓纤维化或消除JAK2突变克隆。此外，JAK抑制剂的持久性或重新激活 在两种MPN小鼠模型中，都观察到JAK/STAT信号在慢性JAK抑制剂治疗中的作用 和MPN患者。因此，迫切需要新的治疗方案来治疗MPN。酪氨酸激酶 RON是MET激酶家族的成员，通过RON发出的信号促进下游的激活 信号，包括PACK、PERK和PSTAT3。RON信号在红细胞中具有独特的作用 增殖和促炎细胞因子的产生。具体地说，构成活跃的短格式RON (SfRON)亚型是Friend病毒通过STAT3信号途径诱导小鼠红白血病所必需的。在……里面 此外，研究还表明，JAK2可以磷酸化标准的全长RON(FlRON)异构体来刺激 红细胞增殖。然而，RON在MPN发病机制中的作用目前尚不清楚。初步数据 我们实验室的研究表明，ALK/MET/RON/ROS1抑制剂Crizotinib对RON的药理抑制作用 抑制患者MPN细胞和JAK2突变细胞系中的克隆形成和JAK/STAT信号转导。 此外，我们还证明了在JAK2突变的细胞中，shRNA敲除了flRON和sfRON两种亚型 LINES表现出了克里佐替尼的抑制作用。我们还发现RON亚型磷酸化是 在JAK抑制剂持久性细胞中增强，提示RON可能增强JAK2持久性表型 对JAK抑制剂的反应。因此，我们假设RON是JAK/STAT信号的一种新的介体 在MPN中，通过RON抑制信号将对MPN患者具有治疗价值。我们建议 利用MPN小鼠模型研究RON信号在MPN疾病进展和启动中的作用。我们 另目的是通过检测MPN在正常对照中的作用来确定靶向RON的治疗窗口。 造血系统。最后，我们建议描述RON增强JAK/STAT的机制 JAK抑制剂NAYVE和JAK抑制剂持久性MPN细胞中的信号转导。

Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. The driver mutations of MPNs, namely activating mutations in either Janus Kinase 2 (JAK2), thrombopoietin receptor (MPL), or calreticulin (CALR), function to upregulate JAK/STAT signaling. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. Currently, the only curative treatment for MPNs is stem cell transplantation, but most patients are poor candidates due to age or comorbidities. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON is a member of the MET kinase family, and signaling through RON promotes activation of downstream signals, including pAkt, pErk, and pSTAT3. RON signaling has well characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. Specifically, the constitutively active short-form RON (sfRON) isoform is necessary for Friend virus-induced erythroleukemia in mice via signaling through STAT3. In addition, it was shown that the canonical full-length RON (flRON) isoform is phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is currently unknown. Preliminary data from our lab has shown that pharmacological inhibition of RON with the ALK/MET/RON/ROS1 inhibitor crizotinib inhibits colony formation and JAK/STAT signaling in both patient MPN cells and JAK2-mutated cell lines. Furthermore, we demonstrated that shRNA knockdown of both flRON and sfRON isoforms in JAK2-mutated cell lines phenocopies the inhibitory effects of crizotinib. We also found that RON isoform phosphorylation is enhanced in JAK inhibitor persistent cells, suggesting that RON may potentiate the JAK2 persistence phenotype in response to JAK inhibitors. Therefore, we hypothesize that RON is a novel mediator of JAK/STAT signaling in MPNs, and that inhibiting signaling through RON will have therapeutic value in MPN patients. We propose to examine the roles of RON signaling in MPN disease progression and initiation using MPN mouse models. We also aim to determine the therapeutic window of targeting RON kinase in MPN by examining its role in the normal hematopoietic system. Lastly, we propose to delineate the mechanism by which RON potentiates JAK/STAT signaling in both JAK inhibitor naïve and JAK inhibitor persistent MPN cells.