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.

摘要 造血干细胞和祖细胞(HSPC)由一个平衡的信号网络调节，这是至关重要的 用于HSPC体内平衡，防止恶变。我们之前展示了这种蛋白质 CBL家族E3泛素连接酶泛素化控制JAK2的稳定性和活性，这对抑制JAK2至关重要 HSPC扩增和髓系恶性肿瘤。在这里，我们发现了一个新的信号轴，其中CBL/JAK2 上调RAB27B以增强NRAS GTPase活性和ERK信号转导。重要的是，异常激活 这一途径对CBL和RAS突变引起的白血病细胞生长至关重要。细胞内信号可以 由调节时间和空间的翻译后修饰(PTM)动态调制 信号蛋白的分布。RAB27B，一个存在于高尔基体和内体中的Rab GTP酶 膜，调节细胞内小泡的运输、对接和与质膜(PM)的融合。Rab27b 基因敲除的小鼠表现出正常的稳态造血。令人惊讶的是，我们发现在初发人群中Rab27b缺乏 HSPC废除突变型，但不取消野生型NRAS介导的信号和细胞生长。从机械上讲，我们 研究表明，RAB27B调节NRAS棕榈酰化、GTP酶活性、稳定性和随后的c- RAF/MEK/ERK激活。RAS蛋白只有在与细胞膜结合时才能传播信号 不同的PTM影响它们在子宫内膜和PM之间的运输的后果因此，a 准确了解RAS与膜的相互作用和运输对于理解RAS的作用是必不可少的 并干预RAS驱动的癌症。RAB27B作为一种新的RAS棕榈酰化调节剂的发现， 对膜锚定的脂类修饰，促使我们进一步定义 CBL/JAK2/RAB27B对RAS/ERK信号的调控及其在恶性肿瘤中的功能意义 HSPC。在目标1中，我们将研究Rab27b缺乏是否可以缓解慢性粒单核细胞白血病(CMML) 突变的NRAS或Cbl缺陷小鼠的发育和恶性HSPC扩增。更重要的是 我们将研究Rab27b缺乏是否会抑制NRAS信号、稳定性、脂质修饰和亚细胞 本地化。在目标2中，我们将使用活细胞成像和生化分析以及遗传方法，以 剖析RAB27B对NRAS转运、棕榈酰化和区域化信号的动态调节。 在目标3中，我们将研究突变的CBL/JAK2在调节RAB27B水平中的作用，并探索其治疗方法 RAB27B在人髓系恶性肿瘤原代HSPC中的靶向潜能。RAS途径突变 包括NRAS和CBL定义的增殖性CMML(PCMML)表型是侵袭性的，易发生 急性髓系白血病转化，并与惨淡的结局相关。这项工作揭示了新的RAB27B介导的 划分的信号动力学对关键信号蛋白至关重要，并为未来奠定基础 分子靶向治疗策略。