MLK3 function in neurofibromatosis tumor cells

MLK3 在神经纤维瘤病肿瘤细胞中的功能

• 批准号: 7088968
• 负责人: John M Kyriakis
• 金额: $ 25.15万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088968
• 关键词: 3T3 cells; RNA interference; SDS polyacrylamide gel electrophoresis; WI38 cell; antisense nucleic acid; cell growth regulation; enzyme activity; enzyme inhibitors; immunoprecipitation; mitogen activated protein kinase; neoplastic cell; neoplastic process; neurofibromatosis; neurofibromatosis type 1 protein /gene; oncogenes; oncoproteins; phosphorylation; protein structure function; serine threonine protein kinase; transcription factor

DESCRIPTION (provided by applicant): The predisposition of type 1 and 2 neurofibromatosis (NF1 and NF2, respectively) patients to both benign and malignant nerve sheath, myeloid cell and other cancers represents for these diseases the principal cause of morbidity and mortality. The effective treatment of these tumors represents a major unmet medical need. NF1 and NF2 are genetic loss of function diseases in which the cognate genes, NF1 and NF2 are subject to a broad suite of inactivating mutations or truncations. Given this genetic heterogeneity, coupled with the loss of function phenotype, targeting or exploiting neurofibromin (the product of NF1) or merlin (the product of NF2) as a therapeutic approach is impractical. By contrast, neurofibromin is a Ras inactivator, and ongoing work indicates that blunting Ras signaling could be beneficial to the treatment of NF1 tumors insofar as inhibition of Ras itself, or inhibition of Ras effectors such as the extracellular signal-regulated kinase (ERK) group of mitogen-activated protein kinases (MAPKs) can significantly blunt NF1 cell proliferation. In a somewhat similar vein, merlin, by an unknown mechanism, suppresses signaling by the Jun-N-terminal kinase (JNK), and possibly the ERK MAPKs. However, the biological consequences of merlin-mediated inhibition of JNK is unclear; and overall, our knowledge of MAPK pathway regulation and function in NF1 and NF2 tumor cell biology is incomplete. Clearly, further studies are needed to identify suitable targets for new treatment approaches. Our preliminary work identifies the Ser/Thr kinase mixed lineage kinase-3 (MLK3) as a required component for the proliferation of malignant schwannoma cell lines from NF1 and NF2 patients, and for murine NF2-/- cells. We find that MLK3 is also required for mitogen activation of NF1/2 cell MAPKs. Surprisingly, MLK3, by an as yet unknown, indirect mechanism, recruits B-Raf to activate ERK. This project will explore the biochemical function(s) that MLK3 performs in mitogen-treated NF tumor cells and the molecular basis by which merlin and neurofibromin regulate MLK.3. Accordingly, in Aim 1 we will use biochemical, pharmacologic, RNAi, morpholino antisense RNA and inducible cell lines to explore (i) if NF2 cell proliferation is ERK and/or JNK-dependent, (ii) if induction of merlin or the NF1 GTPase activating protein-related domain (GRD) inhibits MLK3 and its effectors and if this inhibition is lost in NF2 or NF1 mutants associated with disease, and (iii) how MLK3 regulates ERK-specific MAP3Ks of the Raf family. In Aim 2, we will use RNAi, morpholino antisense RNA and inducible cell lines to assess the degree to which ablation or induction of NF1 or NF2 affects the activity of endogenous MLK3, its effectors and downstream functions. Finally, we find that endogenous and recombinant merlin and MLK3 associate in vivo in a mitogen-reversible manner. Merlin is a negative regulator of JNK activity, and possesses a proline-rich segment which could bind to the SH3 domain of MLK3. Alternatively, merlin could repress the recruitment of MLK3 by Rho family GTPases. In Aim 3, we will use biochemical and molecular biological methods to explore these possibilities.

描述（由申请人提供）：1型和2型神经纤维瘤病（分别为NF1和NF2）患者对良性和恶性神经鞘，髓样细胞和其他癌症均代表了这些疾病的主要原因。这些肿瘤的有效治疗代表了主要的未满足医疗需求。 NF1和NF2是功能疾病的遗传丧失，其中同源基因NF1和NF2受到一系列灭活突变或截断的广泛套件。鉴于这种遗传异质性，再加上功能表型的丧失，靶向或利用神经纤维蛋白（NF1的产物）或Merlin（NF2的产物）作为治疗方法是不切实际的。 By contrast, neurofibromin is a Ras inactivator, and ongoing work indicates that blunting Ras signaling could be beneficial to the treatment of NF1 tumors insofar as inhibition of Ras itself, or inhibition of Ras effectors such as the extracellular signal-regulated kinase (ERK) group of mitogen-activated protein kinases (MAPKs) can significantly blunt NF1 cell proliferation.在某种类似的脉中，梅林通过未知的机制抑制了Jun-N-末端激酶（JNK）以及可能的ERK MAPKS的信号传导。然而，梅林介导的JNK抑制的生物学后果尚不清楚。总体而言，我们对NF1和NF2肿瘤细胞生物学中MAPK途径调节和功能的了解是不完整的。显然，需要进一步的研究来确定适合新治疗方法的目标。我们的初步工作将Ser/THR激酶混合谱系激酶-3（MLK3）视为来自NF1和NF2患者的恶性藻瘤细胞系增殖所需的成分，以及鼠NF2 - / - 细胞。我们发现NF1/2细胞MAPK的有丝分裂原激活也需要MLK3。令人惊讶的是，MLK3通过尚未知道的间接机制，招募B-RAF激活ERK。该项目将探讨MLK3在促丝分裂原处理的NF肿瘤细胞中执行的生化功能以及Merlin和Neurofibromin调节MLK.3的分子基础。 Accordingly, in Aim 1 we will use biochemical, pharmacologic, RNAi, morpholino antisense RNA and inducible cell lines to explore (i) if NF2 cell proliferation is ERK and/or JNK-dependent, (ii) if induction of merlin or the NF1 GTPase activating protein-related domain (GRD) inhibits MLK3 and its effectors and if this inhibition is lost in NF2或NF1突变体与疾病相关，以及（iii）MLK3如何调节RAF家族的ERK特异性MAP3K。在AIM 2中，我们将使用RNAi，Morpholino反义RNA和诱导细胞系评估NF1或NF2的消融或诱导影响内源性MLK3的活性，其效应子和下游功能。最后，我们发现内源性和重组Merlin和MLK3在体内以有丝分裂原的可逆方式助理。 Merlin是JNK活性的负调节剂，并且具有富含脯氨酸的段，该节段可以与MLK3的SH3结构域结合。另外，Merlin可以抑制Rho Family GTPases对MLK3的招募。在AIM 3中，我们将使用生化和分子生物学方法来探索这些可能性。