RON kinase as a novel therapeutic target in myeloproliferative neoplasms

摘要 费城染色体阴性骨髓增生性肿瘤（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 以一个或多个髓系区室的增殖为特征的疾病。驱动突变 MPN，即Janus激酶2（JAK 2）、血小板生成素受体（MPL）或 钙网蛋白（CALR），其功能是上调JAK/STAT信号传导。在疾病进展期间，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，MPN的唯一治愈性治疗是干细胞 移植，但大多数患者是穷人的候选人，由于年龄或合并症。JAK抑制剂ruxolitinib 是一种被批准用于MPN患者的靶向治疗，并已显示出减少脾肿大的能力 以及在患者中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密度。 骨髓纤维化或消除JAK 2突变克隆。此外，JAK抑制剂的持久性或再激活 在两种MPN小鼠模型中均观察到慢性JAK抑制剂治疗后JAK/STAT信号传导的增强 MPN患者因此，MPN迫切需要新的治疗选择。酪氨酸激酶 罗恩是MET激酶家族的成员，并且通过罗恩的信号传导促进下游激酶的活化。 信号，包括pAkt、pErk和pSTAT 3。罗恩信号传导在成红细胞中具有充分表征的作用， 增殖和促炎细胞因子产生。具体而言，组成型活性短型罗恩 （sfRON）同种型是Friend病毒诱导的小鼠红白血病通过STAT 3信号传导所必需的。在 此外，还显示了典型的全长罗恩（fl罗恩）同种型被JAK 2磷酸化，以刺激 成红细胞增殖然而，罗恩在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/罗恩/ROS 1抑制剂克唑替尼对罗恩的药理学抑制作用 抑制患者MPN细胞和JAK 2突变细胞系中的集落形成和JAK/STAT信号传导。 此外，我们证明了JAK 2突变细胞中flRON和sfRON亚型的shRNA敲低， lines表型模仿克唑替尼的抑制作用。我们还发现，罗恩亚型磷酸化是 在JAK抑制剂持续细胞中增强，表明罗恩可能增强JAK 2持续表型 对JAK抑制剂的反应。因此，我们假设罗恩是JAK/STAT信号转导的一种新的介导物 并且通过罗恩抑制信号传导将在MPN患者中具有治疗价值。我们建议 使用MPN小鼠模型检查罗恩信号传导在MPN疾病进展和起始中的作用。我们 还旨在通过检查其在正常人中的作用来确定靶向MPN中罗恩激酶的治疗窗口。 造血系统最后，我们拟阐明罗恩增强JAK/STAT的机制 在JAK抑制剂未处理的和JAK抑制剂持续的MPN细胞中的信号传导。

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.