Novel Regulation of Oncogenic NRAS Signaling in Myeloid Malignancies

髓系恶性肿瘤中致癌 NRAS 信号传导的新调控

• 批准号: 10467363
• 负责人: Wei Tong
• 金额: $ 56.63万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467363
• 关键词: AGFG1 gene; Acute Myelocytic Leukemia; Acyltransferase; Biochemical; Biological Assay; CBL gene; CBLB gene; CD34 gene; Cell Line; Cell membrane; Cells; Cellular Membrane; Chronic Myelomonocytic Leukemia; Collaborations; Cytosol; Data; Development; Disease; Docking; Equilibrium; Family; Future; GOLGA7 gene; Genetic Models; Genetic Transcription; Goals; Golgi Apparatus; Grant; Growth; Guanosine Triphosphate Phosphohydrolases; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Imaging Techniques; JAK2 gene; Knock-in Mouse; Knockout Mice; Leukemic Cell; Link; Lipids; MEKs; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Modification; Molecular; Molecular Target; Mus; Mutate; Mutation; Myeloproliferative disease; Oncogenic; Outcome; Pathway interactions; Patients; Phenotype; Physiological; Post-Translational Protein Processing; Prevention; Prognosis; Proteins; Regulation; Role; Signal Transduction; Signaling Protein; Spatial Distribution; Therapeutic; Ubiquitination; Up-Regulation; Vesicle; Work; cell growth; confocal imaging; cytokine; endosome membrane; genetic approach; in vivo; leukemia; leukemic transformation; live cell imaging; mouse genetics; mutant; novel; palmitoylation; prevent; rab GTP-Binding Proteins; ras Proteins; stem cell growth; stem cell homeostasis; stem cells; trafficking; ubiquitin-protein ligase

Summary Hematopoietic stem and progenitor cells (HSPCs) are regulated by a balanced signaling network, which is critical for HSPC homeostasis and prevention of malignant transformation. We previously showed that protein ubiquitination by CBL family E3 ubiquitin ligases controls JAK2 stability and activity that is important for curbing HSPC expansion and myeloid malignancies. Here we identified a novel signaling axis, where CBL/JAK2 upregulates RAB27B to enhance NRAS GTPase activity and ERK signaling. Importantly, aberrant activation of this pathway is critical for leukemia cell growth conferred by CBL and RAS mutations. Intracellular signaling can be dynamically modulated by post-translational modifications (PTMs) that regulate the temporal and spatial distribution of signaling proteins. RAB27B, a Rab GTPase that is resident in the Golgi and endosome membranes, regulates intracellular vesicle trafficking, docking, and fusion with plasma membrane (PM). Rab27b knockout mice display normal steady-state hematopoiesis. Strikingly, we found that Rab27b deficiency in primary HSPCs abrogates mutant but not wildtype NRAS-mediated signaling and cell growth. Mechanistically, we demonstrated that RAB27B regulates NRAS palmitoylation, GTPase activity, stability, and subsequent c- RAF/MEK/ERK activation. RAS proteins propagate signals only when associated with cellular membranes as a consequence of various PTMs that impact their trafficking between endomembranes and the PM. Therefore, a precise understanding of RAS’ interaction with membranes and trafficking is essential to understand RAS action and to intervene in RAS-driven cancers. The discovery of RAB27B as a novel regulator of RAS palmitoylation, a lipid modification for membrane anchors, propelled us to further define the molecular basis underlying the regulation of RAS/ERK signaling by CBL/JAK2/RAB27B and explore its functional significance in malignant HSPCs. In aim 1, we will investigate if Rab27b deficiency mitigates chronic myelomonocytic leukemia (CMML) development and malignant HSPC expansion induced by mutant Nras or Cbl deficient mice. More importantly we will study if Rab27b deficiency dampens NRAS signaling, stability, lipid modification and subcellular localization. In aim 2, we will use live-cell imaging and biochemical assays as well as genetic approaches, to dissect the dynamic regulation of NRAS trafficking, palmitoylation, and compartmentalized signaling by RAB27B. In aim 3, we will investigate the role of mutant CBL/JAK2 in regulating RAB27B level and explore the therapeutic potential of targeting RAB27B in primary HSPCs from human myeloid malignancies. RAS pathway mutations including NRAS and CBL define the proliferative CMML (pCMML) phenotype that is aggressive, predisposed to AML transformation and associated with dismal outcomes. This work uncovers novel RAB27B-mediated compartmentalized signaling dynamics that is crucial to key signaling proteins, and serves the basis for future therapeutic strategies for molecular targeting.

概括 造血茎和祖细胞（HSPC）受到平衡信号网络的调节，这很关键 用于HSPC稳态和预防恶性转化。我们先前表明蛋白质 CBL家族E3泛素连接酶控制JAK2稳定性和活动对于遏制很重要的泛素化 HSPC扩张和髓样恶性肿瘤。在这里，我们确定了一个新的信号轴，其中CBL/JAK2 上调RAB27B以增强NRAS GTPase活性和ERK信号传导。重要的是，异常激活 该途径对于CBL和RAS突变赋予的白血病细胞生长至关重要。细胞内信号传导可以 通过调节临时和空间的翻译后修饰（PTM）进行动态调节 信号蛋白的分布。 Rab27b，一种Rab GTPase，是高尔基体中的居民 膜，调节细胞内囊泡运输，对接和与质膜（PM）的融合。 rab27b 敲除小鼠表现出正常的稳态造血。令人惊讶的是，我们发现主要的RAB27B缺乏 HSPCS废除了突变体，但没有野生型NRAS介导的信号传导和细胞生长。从机械上讲，我们 证明RAB27B调节NRAS棕榈酰化，GTPase活性，稳定性和随后的C- RAF/MEK/ERK激活。 Ras蛋白仅在与细胞膜相关联时才传播信号 各种PTM的结果影响了它们在内膜和PM之间的贩运。因此， 对RAS与机制和贩运的互动的精确理解对于了解RAS行动至关重要 并干预以RAS驱动的癌症。发现Rab27b是Ras Palmitoylation的新型调节剂， 膜锚的脂质修饰，推动我们进一步定义分子基础 通过CBL/JAK2/RAB27B调节RAS/ERK信号传导，并探索其在恶性中的功能意义 HSPC。在AIM 1中，我们将调查RAB27B缺乏症是否减轻慢性骨髓细胞性白血病（CMML） 突变NRA或CBL缺乏小鼠引起的发育和恶性HSPC扩展。更重要的是 我们将研究RAB27B缺乏症是否会抑制NRAS信号传导，稳定性，脂质修饰和亚细胞 本土化。在AIM 2中，我们将使用活细胞成像和生化测定以及遗传方法 解剖Rab27b的NRAS运输，棕榈酰化和分隔的信号的动态调节。 在AIM 3中，我们将研究突变CBL/JAK2在调节RAB27B水平并探索疗法的作用 来自人髓样恶性肿瘤中原代HSPC中RAB27B的潜力。 RAS途径突变 包括NRA和CBL定义了侵略性的增殖剂CMML（PCMML）表型 AML转换并与惨淡的结果相关。这项工作揭示了新颖的Rab27b介导的 对关键信号蛋白至关重要的隔室化信号传导动力学，并为将来提供基础 分子靶向的治疗策略。