Targeting allosteric scaffolding functions of Aurora kinase A in cancer

摘要 神经母细胞瘤是婴儿最常见的实体瘤。大约25%的患者有高风险 神经母细胞瘤是一种预后不良且治疗选择很少的毁灭性疾病。的主要驱动力 高危神经母细胞瘤是癌基因MYCN，一种MYC家族转录因子， 口袋里，并长期逃避药物开发的努力。 最近，蛋白激酶Aurora A（AurA）被证明与神经母细胞瘤中的N-Myc蛋白结合 细胞，并通过SCF泛素连接酶复合物干扰其泛素化，阻止N-Myc被 被蛋白体降解阻断AurA和N-Myc之间的复合物形成导致快速的N-Myc 神经母细胞瘤细胞系中的降解和细胞死亡。同样的AurA/N-Myc复合物现在已经被证明 驱动神经内分泌前列腺癌（NEPC），而AurA也形成了一个类似的复合物， 相关c-Myc蛋白在肝癌中的表达。这些最近的发现指出了靶向Myc的新范例， 家族转录因子在癌症中使用抑制剂，触发AurA的结构变化， Myc蛋白结合并促进Myc降解。 我们的实验室最近表明，大多数现有的AurA抑制剂，包括目前的临床候选药物， alisertib对AurA没有足够强的变构作用，不能有效削弱N-Myc结合。在 与此一致，alisertib对细胞系中N-Myc水平的影响不一致，并且表现不佳 正在进行的神经母细胞瘤和NEPC的临床试验。我们目前对极光如何 与c-Myc和N-Myc结合，这些相互作用如何受到抑制剂的影响是一个主要障碍 这种针对Myc驱动的癌症的治疗策略。 这个项目的目标是提供缺失的分子之间的相互作用的图片 AurA和Myc转录因子以及它们如何通过抑制剂结合来调节。我们计划使用 新的实验工具和方法，以确定如何结合的c-Myc和N-Myc改变构象 研究AurA的结构（形状）和动力学（蛋白质运动），并描述抑制剂的特定结构变化， 必须触发以有效地使这些复合物不稳定。我们将a）定义AurA/Myc的结构 使用X射线晶体学、磁共振光谱学和分子光谱学 建模，B）通过跟踪关键结构确定这些相互作用如何改变AurA构象和动力学 c）将一大组激酶抑制剂对AurA的影响关联起来 d）测试它们的构象与它们改变N-Myc和c-Myc的结合亲和力的能力，和 在一系列N-Myc和c-Myc依赖性癌细胞系中最强的AurA变构调节剂， 神经母细胞瘤、NEPC和肝癌细胞。这些见解将为重新利用现有的 激酶抑制剂和开发新的抑制剂作为Myc驱动的癌症的新治疗方式。

ABSTRACT Neuroblastoma is the most common solid tumor in infants. About 25% of patients have high-risk neuroblastoma, a devastating disease with poor prognosis and few treatment options. The primary driver of high-risk neuroblastoma is the oncogene MYCN, a MYC-family transcription factor that has no druggable pockets and has long eluded drug development efforts. Recently, the protein kinase Aurora A (AurA) was shown to bind to the N-Myc protein in neuroblastoma cells and interfere with its ubiquitination by the SCF ubiquitin ligase complex, preventing N-Myc from being degraded by the proteosome. Blocking complex formation between AurA and N-Myc results in rapid N-Myc degradation and cell death in neuroblastoma cell lines. The same AurA/N-Myc complex has now been shown to drive neuroendocrine prostate cancer (NEPC), and AurA also forms a similar complex with the closely- related c-Myc protein in liver cancer. These recent discoveries point to a new paradigm for targeting Myc- family transcription factors in cancer using inhibitors that trigger structural changes in AurA that block Myc protein binding and promote Myc degradation. Our lab has recently shown that most existing AurA inhibitors, including the current clinical candidate alisertib, do not have a strong enough allosteric effect on AurA to be effective at weakening N-Myc binding. In agreement with this, alisertib has inconsistent effects on N-Myc levels in cell lines, and has not performed well in ongoing clinical trials in neuroblastoma and NEPC. The weakness in our current understanding of how AurA binds to c-Myc and N-Myc and how these interactions are affected by inhibitors represents a major impediment to this therapeutic strategy for targeting Myc-driven cancers. The goal of this project is to provide the missing molecular picture of the interactions between AurA and Myc transcription factors and how they can be modulated by inhibitor binding. We plan to use new experimental tools and approaches to define how the binding of c-Myc and N-Myc alters the conformation (shape) and dynamics (protein motion) of AurA, and to delineate the specific structural changes an inhibitor must trigger to efficiently destabilize these complexes. We will a) define the structure of the AurA/Myc complexes at atomic resolution using x-ray crystallography, magnetic resonance spectroscopies and molecular modeling, b) determine how these interactions alter AurA conformation and dynamics by tracking key structural elements of the kinase in solution, c) correlate the effects of a large panel of kinase inhibitors on AurA conformation with their ability to alter the binding affinities of N-Myc and c-Myc, and d) test the efficiency of the strongest AurA allosteric modulators in a series of N-Myc- and c-Myc-dependent cancer cell lines including neuroblastoma, NEPC and liver cancer cells. The insights will pave the way for the repurposing of existing kinase inhibitors and the development of new inhibitors as a new treatment modality for Myc-driven cancers.