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。约25%的患者患有高危疾病 神经母细胞瘤是一种毁灭性的疾病，预后很差，几乎没有治疗选择。其主要驱动力是 高危神经母细胞瘤是癌基因MYCN，是一种MYC家族转录因子，没有可药物 他们中饱私囊，长期以来一直逃避药物开发努力。 最近，在神经母细胞瘤中，蛋白激酶Aurora A(Aura)被发现与N-Myc蛋白结合 并通过SCF泛素连接酶复合体干扰其泛素化，阻止N-Myc被 被蛋白质小体降解。阻断AURA和N-Myc之间的复合体形成导致快速N-Myc 神经母细胞瘤细胞系的降解和细胞死亡。现在已经显示了相同的AURA/N-Myc复合体 以推动神经内分泌前列腺癌(NEPC)，而AURA也形成了类似的复合体，与密切相关的- 相关c-Myc蛋白在肝癌中的表达这些最新的发现指出了一种针对Myc的新范式- 癌症中的家族转录因子使用抑制剂触发先兆的结构变化，从而阻断 结合MYC蛋白，促进Myc降解。 我们的实验室最近显示，大多数现有的AURA抑制剂，包括目前的临床候选药物 Alisertib对AURA没有足够强的变构效应来有效地削弱N-Myc结合。在……里面 与此一致的是，alisertib对细胞株中N-Myc水平的影响不一致，而且表现不佳 正在进行的神经母细胞瘤和NEPC的临床试验中。我们目前对气场的理解存在弱点 结合c-Myc和N-Myc，这些相互作用如何受到抑制剂的影响是一个主要障碍 这一针对Myc驱动的癌症的治疗策略。 这个项目的目标是提供缺少的相互作用的分子图像。 AURA和Myc转录因子以及它们如何受抑制物结合的调节。我们计划使用 确定c-Myc和N-Myc结合如何改变构象的新实验工具和方法 (形状)和动力学(蛋白质运动)，并描绘特定的结构变化的抑制剂 必须触发才能有效地破坏这些复合体的稳定。我们将a)定义AURA/Myc的结构 用X-射线结晶学、磁共振波谱和分子光谱研究原子分辨率的配合物 建模，b)通过跟踪关键的结构来确定这些相互作用如何改变灵气构象和动力学 溶液中的激酶成分，c)与一大批激酶抑制剂对先兆的影响相关。 与它们改变N-Myc和c-Myc结合亲和力的能力的构象，以及d)测试 一系列N-Myc和c-Myc依赖的癌细胞系中最强的AURA变构调节剂，包括 神经母细胞瘤、NEPC和肝癌细胞。这些见解将为现有的 激酶抑制剂和作为Myc驱动的癌症新治疗方式的新抑制剂的开发。

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.