MLK3 function in neurofibromatosis tumor cells

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、髓细胞和其他癌症的易感性是这些疾病发病率和死亡率的主要原因。这些肿瘤的有效治疗是一个重大的未得到满足的医疗需求。NF1和NF2是一种遗传性功能丧失疾病，在这些疾病中，同源基因NF1和NF2会受到一系列失活突变或截断的影响。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)作为一种治疗方法是不切实际的。相反，神经纤维蛋白是一种RAS失活剂，正在进行的研究表明，钝化RAS信号可能有利于NF1肿瘤的治疗，因为抑制RAS本身，或抑制RAS效应，如细胞外信号调节激酶(ERK)组的丝裂原活化蛋白激酶(MAPKs)可以显著抑制NF1细胞的增殖。在某种程度上，Merlin以一种未知的机制抑制Jun-N末端激酶(JNK)的信号传递，可能还有ERK MAPK的信号传递。然而，Merlin介导的抑制JNK的生物学后果尚不清楚；总的来说，我们对MAPK通路在NF1和NF2肿瘤细胞生物学中的调控和功能的了解还不完整。显然，需要进一步的研究来确定新的治疗方法的合适靶点。我们的初步工作确定丝氨酸/苏氨酸激酶混合谱系激酶-3(MLK3)是来自NF1和NF2患者的恶性神经鞘瘤细胞系以及小鼠NF2-/-细胞增殖所必需的成分。我们发现MLK3也是NF1/2细胞MAPK有丝分裂原激活所必需的。令人惊讶的是，MLK3通过一种未知的间接机制招募B-Raf来激活ERK。本项目将探索MLK3在有丝分裂原处理的NF肿瘤细胞中发挥的生化功能(S)，以及Merlin和神经纤维蛋白调控MLK3的分子基础。因此，在目标1中，我们将使用生化、药理学、RNAi、吗啡反义RNA和可诱导细胞系来探索(I)NF2细胞的增殖是否依赖ERK和/或JNK，(Ii)诱导Merlin或NF1 GTPase激活蛋白相关结构域(GRD)是否抑制MLK3及其效应器，以及这种抑制是否在与疾病相关的NF2或NF1突变体中丢失，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、吗啡反义RNA和可诱导细胞系来评估消融或诱导NF1或NF2对内源性MLK3的活性、其效应和下游功能的影响程度。最后，我们发现内源性和重组的Merlin和MLK3在体内以丝裂原可逆的方式结合。Merlin是JNK活性的负调控因子，具有一个富含Pro的片段，可与MLK3的SH3结构域结合。或者，Merlin可以抑制Rho家族GTP酶对MLK3的招募。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。

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.