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: 7224176
• 负责人: John M Kyriakis
• 金额: $ 24.42万
• 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/7224176
• 关键词: Ablation; Affect; Antisense RNA; Benign; Binding; Biochemical; Biological; Cell Line; Cell Proliferation; Cells; Coupled; Disease; Extracellular Signal Regulated Kinases; Family; GTPase-Activating Proteins; Genetic; Genetic Heterogeneity; Guanosine Triphosphate Phosphohydrolases; Heterogeneity; Knowledge; Malignant - descriptor; Malignant Neoplasms; Malignant Peripheral Nerve Sheath Tumor; Mediating; Medical; Methods; Mitogen-Activated Protein Kinases; Mitogens; Molecular; Morbidity - disease rate; Mus; Mutation; Myeloid Cells; N-terminal; NF1 gene; Nerve Sheaths; Neurofibromatoses; Neurofibromatosis 2; Neurofibromatosis Type 1 Protein; Neurofibromin 2; Pathway interactions; Patients; Phenotype; Phosphotransferases; Physiological; Predisposition; Proline; RNA Interference; Recombinants; Recruitment Activity; Regulation; SH3 Domains; Signal Transduction; Therapeutic; Tumor Cell Biology; Veins; Work; base; in vivo; inhibitor/antagonist; loss of function; mixed lineage kinase 3; mortality; mutant; neoplastic cell; rho; stress-activated protein kinase 1; tumor

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、骨髓细胞和其他癌症的易感性是这些疾病发病和死亡的主要原因。这些肿瘤的有效治疗代表了一个重大的未满足的医疗需求。 NF1 和 NF2 是遗传性功能丧失疾病，其中同源基因 NF1 和 NF2 遭受广泛的失活突变或截短。鉴于这种遗传异质性，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。相比之下，神经纤维蛋白是一种 Ras 灭活剂，正在进行的工作表明，抑制 Ras 本身或抑制 Ras 效应物（例如丝裂原激活蛋白激酶 (MAPK) 的细胞外信号调节激酶 (ERK) 组）可以显着抑制 NF1 细胞增殖，因此削弱 Ras 信号传导可能有利于治疗 NF1 肿瘤。与此类似，merlin 通过未知机制抑制 Jun-N 末端激酶 (JNK) 以及可能的 ERK MAPK 的信号传导。然而，merlin 介导的 JNK 抑制的生物学后果尚不清楚。总的来说，我们对 NF1 和 NF2 肿瘤细胞生物学中 MAPK 通路调节和功能的了解并不完整。显然，需要进一步研究来确定新治疗方法的合适靶点。我们的初步工作确定 Ser/Thr 激酶混合谱系激酶 3 (MLK​​3) 是 NF1 和 NF2 患者的恶性神经鞘瘤细胞系以及小鼠 NF2-/- 细胞增殖所需的成分。我们发现 MLK3 也是 NF1/2 细胞 MAPK 丝裂原激活所必需的。令人惊讶的是，MLK3 通过一种迄今未知的间接机制招募 B-Raf 来激活 ERK。该项目将探索 MLK3 在丝裂原处理的 NF 肿瘤细胞中发挥的生化功能，以及 merlin 和神经纤维蛋白调节 MLK.3 的分子基础。因此，在目标 1 中，我们将使用生化、药理学、RNAi、吗啉代反义 RNA 和诱导细胞系来探索 (i) NF2 细胞增殖是否依赖于 ERK 和/或 JNK，(ii) merlin 或 NF1 GTPase 激活蛋白相关结构域 (GRD) 的诱导是否会抑制 MLK3 及其效应子，以及这种抑制是否在 与疾病相关的 NF2 或 NF1 突变体，以及 (iii) MLK3 如何调节 Raf 家族的 ERK 特异性 MAP3K。在目标 2 中，我们将使用 RNAi、吗啉代反义 RNA 和诱导型细胞系来评估 NF1 或 NF2 的消融或诱导对内源性 MLK3、其效应子和下游功能的活性的影响程度。最后，我们发现内源重组 merlin 和 MLK3 在体内以有丝分裂原可逆的方式结合。 Merlin 是 JNK 活性的负调节因子，并拥有富含脯氨酸的片段，可以与 MLK3 的 SH3 结构域结合。或者，merlin 可以抑制 Rho 家族 GTPases 招募 MLK3。在目标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。