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.

总结