IL-1 Receptor-Associated Kinase as a Cancer Therapeutic Target

描述（由申请人提供）： 在过去的30年中，黑色素瘤的发病率一直在上升，转移性黑色素瘤患者的5年生存率接近2%;退伍军人患黑色素瘤的可能性是普通人群的4.5倍。这些令人沮丧的统计数据部分是由于黑色素瘤对癌症治疗的耐药性是众所周知的。我们报道了黑色素瘤细胞系和患者样本表达升高水平的IL-1受体相关激酶-4（IRAK-4）和IRAK-1。IRAK-4在Toll样受体、IL-1、IL-18和IL-33信号传导中起中心作用。用药理学抑制剂或siRNA减弱黑色素瘤细胞中的IRAK-1，-4信号传导减少了已知促进血管生成和诱导肿瘤相关成纤维细胞的各种趋化因子/细胞因子的产生。IRAK抑制还增强了某些化学疗法在体外和荷瘤小鼠中的作用。相反，黑色素瘤细胞中IRAK-4信号传导的激活增加了这些细胞因子/趋化因子的产生并促进了化疗抗性。来自过表达IRAK-4基因的肿瘤细胞的上清液增强了内皮细胞增殖并激活了人成纤维细胞，如通过腱生蛋白-C和<$-SMA的表达增加所证明的。有趣的是，虽然已知IRAK-4定位于细胞溶质中，但我们观察到在黑素瘤中IRAK-4易位至细胞核，并且这在化疗暴露后增加。总体假设是IRAK-4核动员促进化学抗性，并且IRAK信号传导协调肿瘤微环境的变化，促进肿瘤生长和转移。拟议的研究重点是获得关于肿瘤细胞中IRAK-4信号传导如何的更大机制见解（分子和细胞）：（1）影响血管生成，化疗耐药性和转移，（2）改变肿瘤微环境，（3）可用作治疗靶点以增强化疗的疗效。目的1是获得关于核IRAK-4在化疗抗性和基因表达中所起作用的机制理解。确定核IRAK-4的功能是新颖的，并且独立于已知的IRAK-4介导的激活机制。这将使用经工程改造以表达IRAK-4变体（包括野生型、shRNA-IRAK、激酶死亡IRAK和IRAK- 4 SUMO）的黑色素瘤细胞系进行研究，其中SUMO化位点已突变以增加胞质水平但防止核动员。目的2是确定抑制黑色素瘤中的IRAK-4对转移和血管生成的影响。我们将使用小鼠（BRAFV 600 PTEN小鼠）中的黑色素瘤的诱导型模型以及被工程化以过表达或敲低IRAK-4的人和小鼠黑色素瘤细胞，以使用体内和离体模型监测肿瘤新血管形成和转移。目的3是发现黑色素瘤中IRAK-4信号在改变肿瘤微环境以促进肿瘤生长中的作用。基于初步数据显示黑色素瘤中的IRAK-4信号传导诱导支持肿瘤相关成纤维细胞的因子的表达，该目标将探索细胞机制并研究先前未被认识的概念，即肿瘤细胞内的IRAK信号传导对于协调有利于肿瘤生长和转移的微环境的产生至关重要。目的4寻求确定当与化学疗法组合时靶向递送IRAK抑制剂至肿瘤的治疗功效。我们将使用抗PD-L1抗体来递送FDA批准的化疗药物加上IRAK-1，-4抑制剂（目前处于II/III期临床试验中）。这些研究的结果将为黑色素瘤中的IRAK-4信号传导如何增强化疗耐药性和转移提供更深入的分子和细胞理解，并将确定新的治疗靶点以增强化疗反应。

DESCRIPTION (provided by applicant): The incidence of melanoma has been on the rise over the last 30 years with the 5 year survival rate approaching 2% for patients with metastatic melanoma; veterans are up to 4.5 times more likely to develop melanoma than the general population. These dismal statistics are partly due to the fact that melanoma is notoriously resistant to cancer therapies. We reported that melanoma cell lines and patient samples express elevated levels of the IL-1 receptor-associated kinase-4 (IRAK-4) and IRAK-1. IRAK-4 plays a central role in Toll-like receptor, IL-1, IL-18 and IL-33 signaling. Attenuating IRAK-1,-4 signaling in melanoma cells with pharmacological inhibitors or siRNA diminished the production of various chemokines/cytokines known to promote angiogenesis and the induction of tumor-associated fibroblasts. IRAK inhibition also enhanced the effects of certain chemotherapies in vitro and in tumor-bearing mice. In contrast, the activation of IRAK-4 signaling in melanoma cells increased the production of these cytokines/chemokines and promoted chemotherapeutic resistance. Supernatant from tumor cells engineered to overexpress the IRAK-4 gene augmented endothelial cell proliferation and activated human fibroblasts, as demonstrated by increased expression of Tenascin-C and ¿-SMA. Interestingly, while IRAK-4 is known to localize in the cytosol, we observed that in melanoma IRAK-4 translocates to the nucleus and this is increased upon chemotherapy exposure. The overarching hypothesis is that IRAK-4 nuclear mobilization promotes chemoresistance and that IRAK signaling orchestrates changes in the tumor microenvironment that promotes tumor growth and metastases. The proposed studies are focused on gaining greater mechanistic insights (molecular and cellular) on how IRAK-4 signaling in tumor cells: (1) influences angiogenesis, chemoresistance and metastases, (2) changes the tumor microenvironment, and (3) can be used as a therapeutic target to enhance the efficacy of chemotherapies. Aim 1 is to gain a mechanistic understanding regarding the role that nuclear IRAK-4 plays in chemotherapy resistance and gene expression. Determining the function of nuclear IRAK-4 is novel and independent of known mechanisms of IRAK-4-mediated activation. This will be investigated using melanoma lines engineered to express IRAK-4 variants including wild type, shRNA-IRAKs, kinase dead IRAKs, and IRAK- 4 SUMO, in which SUMOylation sites have been mutated to increase cytosolic levels but prevent nuclear mobilization. Aim 2 is to determine the effect that inhibiting IRAK-4 in melanoma has on metastasis and angiogenesis. We will use an inducible model of melanoma in mice (BRAFV600PTEN mice) as well as human and mouse melanoma cells engineered to overexpress or knockdown IRAK-4 to monitor tumor neovascularization and metastases using in vivo and ex vivo models. Aim 3 is to discover the role that IRAK-4 signaling in melanoma plays in altering the tumor microenvironment to promote tumor growth. Building on preliminary data showing that IRAK-4 signaling in melanoma induces the expression of factors that support tumor associated fibroblasts, this aim will explore the cellular mechanism and investigate the previously unrecognized concept that IRAK signaling within tumor cells is critical for orchestrating the generation of a microenvironment favoring tumor growth and metastasis. Aim 4 seeks to determine the therapeutic efficacy of targeted delivery of IRAK inhibitor to tumors when combined with chemotherapies. We will use anti-PD-L1 antibodies to deliver FDA-approved chemotherapies plus IRAK-1,-4 inhibitor (currently in Phase II/III clinical trials). The results from these studies will provide a greater molecular and cellular understanding of how IRAK-4 signaling in melanoma enhances chemoresistance and metastasis and will identify new therapeutic targets to enhance chemotherapeutic responses.