Novel Regulation of Oncogenic NRAS Signaling in Myeloid Malignancies

总结 造血干细胞和祖细胞（HSPCs）受平衡的信号网络调节，这是至关重要的 用于HSPC稳态和预防恶性转化。我们之前发现这种蛋白质 CBL家族E3泛素连接酶的泛素化控制JAK 2的稳定性和活性，这对于抑制JAK 2的表达是重要的。 HSPC扩增和骨髓恶性肿瘤。在这里，我们确定了一个新的信号传导轴，其中CBL/JAK 2 上调RAB 27 B以增强NRAS GT3活性和ERK信号传导。重要的是， 该途径对于CBL和RAS突变所赋予的白血病细胞生长是关键的。细胞内信号可以 通过调节时间和空间的翻译后修饰（PTM）进行动态调节 信号蛋白的分布。RAB 27 B，一种存在于高尔基体和内体中的Rab GTdR 膜，调节胞内囊泡运输，对接和融合与质膜（PM）。Rab27b 敲除小鼠显示正常稳态造血。令人惊讶的是，我们发现Rab 27 b缺乏症在原发性 HSPC消除突变体而非野生型NRAS介导的信号传导和细胞生长。机械地，我们 证明了RAB 27 B调节NRAS棕榈酰化、GT3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传播信号， 影响其在内膜和PM之间运输的各种PTM的后果。因此 精确理解RAS与膜的相互作用和运输对于理解RAS的作用是至关重要的 并干预RAS引发的癌症。RAB 27 B作为RAS棕榈酰化的新型调节剂的发现， 膜锚的脂质修饰，促使我们进一步确定膜锚的分子基础。 CBL/JAK 2/RAB 27 B对RAS/ERK信号通路的调控及其在恶性肿瘤中的作用 HSPC。在目标1中，我们将研究Rab 27 b缺陷是否减轻慢性粒单核细胞白血病（CMML） 突变型Nras或Cbl缺陷型小鼠诱导的HSPC发育和恶性扩增。更重要的 我们将研究Rab 27 b缺陷是否会抑制NRAS信号传导、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， 剖析NRAS运输、棕榈酰化和由RAB 27 B区室化信号传导的动态调节。 目的3：研究突变型CBL/JAK 2在调节RAB 27 B水平中的作用，并探讨其治疗作用。 靶向来自人骨髓恶性肿瘤的原发性HSPC中的RAB 27 B的潜力。RAS通路突变 包括NRAS和CBL在内的增殖性CMML（pCMML）表型是侵袭性的，易患 AML转化并伴有令人沮丧的结局。这项工作揭示了新型RAB 27 B介导的 区室化的信号动力学对关键信号蛋白至关重要，并为未来的研究奠定基础。 分子靶向的治疗策略。

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.