Identification of DCLK2-TBK1 signaling axis as a potential therapeutic target in kidney cancer

项目摘要 肾透明细胞癌(CcRCC)约占所有肾癌的85%，是耐药的。 可用于多种癌症疗法，具有极高的致命性。Von Hippel-Lindau(VHL)是最重要的肿瘤 肾癌中的抑制物，其缺失导致缺氧诱导因子α(α，包括hif1α和hif2α) 积累。尽管HIF2α抑制剂已被开发为肾癌的潜在治疗靶点， 只有一小部分患者对α抑制剂治疗有反应，这表明 识别VHL缺陷性肾癌的额外治疗脆弱性。我们最近出版的作品 研究表明，VHL的丢失激活了在肿瘤细胞中起关键作用的坦克结合激酶1(TBK1) 增殖、肿瘤生长和自发转移。靶向TBK1抑制VHL基因缺陷的癌细胞 在保持VHL恢复细胞不受影响的情况下生长。而TBK1参与先天免疫信号和 使转录因子IRF3磷酸化，我们没有在VHL中观察到p-IRF3(或I型干扰素的产生)。 肿瘤缺陷提示在ccRCC中TBK1的功能交替。因此，TBK1是一种很有前途的治疗方法 目标。然而，目前可用的抑制剂缺乏特异性，可能会损害先天免疫。身份识别 CcRCC中的TBK1调节因子可能导致开发出更特异的抑制物，从而在 其他隔膜(即免疫细胞)。通过全基因组siRNA筛选，我们鉴定出双重皮质醇类似 激酶2(DCLK2)是一种潜在的TBK1激活剂。DCLK2被多个siRNAs耗尽降低了TBK1 磷酸化和蛋白质水平。反之，DCLK2的过表达诱导了TBK1的磷酸化 在体外和体内。虽然DCLK2被报道促进视神经损伤后生长锥的重建，但一种 DCLK2在癌症中的作用尚不清楚。我们的初步数据显示，DCLK2耗尽减少了ccRCC细胞 增殖、集落形成和异种移植瘤生长。我们假设DCLK2-TBK1调控 在伴有VHL缺失的肾小管上皮细胞癌中，Node是一种新的信号轴，可用于治疗。 这是第一次研究DCLK2在癌症中的作用。此外，TBK1是第一个已知的DCLK2 癌症中的激酶底物。在特定的目标1中，我们将描述DCLK2在肾脏中的功能意义 癌症。在特定的目标2中，我们将描述DCLK2对肾脏的贡献的分子机制 肿瘤发生学。在特定的目标3中，我们将研究靶向DCLK2-TBK1在肾脏中的治疗潜力 可通过UT西南肾获得的癌症原位异种移植和患者来源的异种移植(PDX) 癌症卓越研究专项计划(孢子)。成功完成这些目标将提供 对一种新的信号通路的机械性洞察并为靶向治疗干预奠定基础 CcRCC中的DCLK2-TBK1轴。

Project Summary Clear cell renal cell carcinoma (ccRCC), which accounts for approximately 85% of all renal cancers, is resistant to a variety of cancer therapies and is highly lethal. von Hippel-Lindau (VHL) is the most important tumor suppressor in renal cancer, its loss leads to hypoxia inducible factor α (HIFα, including HIF1α and HIF2α) accumulation. Although HIF2α inhibitor has been developed as a potential therapeutic target in renal cancer, only a small propotion of patients will respond to HIF2α inhibitore treatment, suggesting the importance of identifying additional therapeutic vulnerabilities in VHL-deficient kidney cancer. Our recently published work demonstrated that VHL loss activates TANK Binding Kinase 1 (TBK1), which plays a critical role in tumor cell proliferation, tumor growth and spontaneous metastasis. Targeting TBK1 inhibits VHL-deficient cancer cell growth while leaving VHL restored cells unaffected. While TBK1 is involved in innate immune signaling and phosphorylates the transcription factor IRF3, we did not observe p-IRF3 (or type I interferon production) in VHL- deficient cancers suggesting alternate functions for TBK1 in ccRCC. Thus, TBK1 is a promising therapeutic target. However, currently available inhibitors lack specificity and may impair innate immunity. Identification of TBK1 regulators in ccRCC may lead to development of more specific inhibitors that preserve TBK1 function in other compartments (i.e. immune cells). Through a kinome-wide siRNA screen, we identified Doublecortin Like Kinase 2 (DCLK2) as a potential TBK1 activator. DCLK2 depletion by multiple siRNAs decreased TBK1 phosphorylation and protein levels. Conversely, overexpression of DCLK2 induced TBK1 phosphorylation both in vitro and in vivo. While DCLK2 was reported to promote growth cone reformation after optic nerve injury, a role for DCLK2 in cancer is unexplored. Our preliminary data show that DCLK2 depletion reduced ccRCC cell proliferation, colony formation and xenograft tumor growth. We hypothesize that the DCLK2-TBK1 regulatory node serves as a novel signaling axis in ccRCCs with VHL loss that could be exploited therapeutically. This is the first study to characterize the role of DCLK2 in cancer. In addition, TBK1 is the first known DCLK2 kinase substrate in cancer. In Specific Aim 1, we will characterize the functional significance of DCLK2 in kidney cancer. In Specific Aim 2, we will delineate the molecular mechanism by which DCLK2 contributes to kidney tumorigenesis. In Specific Aim 3, we will investigate the therapeutic potential of targeting DCLK2-TBK1 in kidney cancer ortothopic xenografts and patient derived xenografts (PDXs) available through UT Southwestern Kidney Cancer Specialized Program of Research Excellence (SPORE). Successful completion of these aims will provide mechanistic insight into a novel signaling pathway and set the foundation for therapeutic interventions targeting the DCLK2-TBK1 axis in ccRCC.