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: 10580053
• 负责人: Wei Tong
• 金额: $ 53.64万
• 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/10580053
• 关键词: AGFG1 gene; Acute Myelocytic Leukemia; Acyltransferase; Biochemical; Biological Assay; CBL gene; CD34 gene; Cell Line; Cell membrane; Cells; Cellular Membrane; Chronic Myelomonocytic Leukemia; Collaborations; Cytosol; Data; Development; Disease; Docking; Family; Future; GOLGA7 gene; Genetic Models; Genetic Transcription; Goals; 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; 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; RAS driven cancer; Regulation; Role; Signal Transduction; Signaling Protein; Spatial Distribution; Therapeutic; Ubiquitination; Up-Regulation; Vesicle; Work; Xenograft procedure; 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 造血干细胞和祖细胞 (HSPC) 由平衡的信号网络调节，这一点至关重要 我们之前证明了蛋白质对于 HSPC 稳态和预防恶性转化的作用。 CBL 家族 E3 泛素连接酶的泛素化控制 JAK2 的稳定性和活性，这对于抑制 HSPC 扩张和骨髓恶性肿瘤。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 GTP 酶活性和 ERK 信号传导，重要的是，异常激活。 该通路对于 CBL 和 RAS 突变导致的白血病细胞生长至关重要。 通过调节时间和空间的翻译后修饰（PTM）进行动态调节 RAB27B 是一种存在于高尔基体和内体中的 Rab GTP 酶。 膜，调节细胞内囊泡运输、对接以及与质膜 (PM) 的融合。 令人惊讶的是，我们发现原代小鼠存在 Rab27b 缺陷。 从机制上讲，我们发现 HSPC 会消除突变型 NRAS 介导的信号传导和细胞生长，但不会消除野生型 NRAS 介导的信号传导和细胞生长。 RAB27B 调节 NRAS 棕榈酰化、GTP 酶活性、稳定性和随后的 c- RAF/MEK/ERK 激活仅在与细胞膜相关时传播信号。 影响内膜和 PM 之间运输的各种 PTM 的结果。 准确理解 RAS 与膜和运输的相互作用对于理解 RAS 的作用至关重要 并干预 RAS 驱动的癌症，发现 RAB27B 作为 RAS 棕榈酰化的新型调节剂， 对膜锚的脂质修饰，促使我们进一步定义其分子基础 CBL/JAK2/RAB27B对RAS/ERK信号通路的调控及其在恶性肿瘤中的功能意义 在目标 1 中，我们将研究 Rab27b 缺乏是否可以减轻慢性粒单核细胞白血病 (CMML)。 更重要的是，Nras 或 Cbl 缺陷小鼠诱导的发育和恶性 HSPC 扩增。 我们将研究 Rab27b 缺陷是否会抑制 NRAS 信号传导、稳定性、脂质修饰和亚细胞 在目标 2 中，我们将使用活细胞成像和生化检测以及遗传方法来定位。 剖析 RAB27B 对 NRAS 运输、棕榈酰化和区室化信号传导的动态调节。 在目标3中，我们将研究突变体CBL/JAK2在调节RAB27B水平中的作用并探索治疗方法 靶向RAB27B在人类骨髓恶性肿瘤原发性HSPC中的潜力。 包括 NRAS 和 CBL 定义了增殖性 CMML (pCMML) 表型，该表型具有侵袭性、易于 这项工作揭示了新的 RAB27B 介导的 AML 转化及其相关结果。 分隔的信号动力学对于关键信号蛋白至关重要，并为未来奠定了基础 分子靶向治疗策略。