Functional impact, mechanistic role, and targetability of ROS1 aberrations in cancer

项目摘要 原癌基因ROS 1的染色体重排产生组成型活性ROS 1激酶融合 这些蛋白质被确定为人类癌症中的可药用致病驱动因子。目前，这是唯一的 证实了肿瘤发生中ROS 1异常激活的机制。ROS 1可与多种酪氨酸靶向 激酶抑制剂（TKI），并在ROS 1融合阳性患者中观察到显著的肿瘤消退， 使用靶向TKI治疗。癌症基因组测序研究揭示了许多ROS 1体细胞突变， 它们对催化功能的影响尚未得到测试，这引发了关于ROS 1驱动的癌症的新问题 发病机制此外，ROS 1羧基末端在控制细胞内或细胞外的细胞内代谢中的机制作用也被阐明。 分子间调节是未知的。从广义上讲，这些知识差距，关于监管的区域和次区域办事处1 以及其结构-功能关系，阻碍了积累的癌症基因组数据的有意义的利用。 我们的总体假设是，ROS 1酪氨酸激酶和羧基末端结构域的表征， 体细胞突变的透镜将揭示受体调节的生物学基础，并有助于 癌症基因组数据的翻译。我们将通过回答以下问题来实验性地解决这个假设 问题： (1)癌症相关的ROS 1酪氨酸激酶结构域（TKD）突变是否激活催化功能， 这是否足以单独或协同进行肿瘤转化或转移性播散 与热点癌蛋白有关吗初步数据提供了设计的活化ROS 1 TKD 突变转化细胞，并且可以用ROS 1-TKI靶向。我们将测试20个优先体细胞ROS 1 TKD 突变增强催化功能，诱导细胞转化和肿瘤形成， 敏感性模式 (2)ROS 1的羧基末端结构域（CTD）如何调节蛋白质稳定性和TKI诱导的 蛋白质下调？我们的试验数据表明，（a）ROS 1是泛素化的，（B）催化失活的ROS 1 经历蛋白酶体辅助降解，和（c）可能涉及ROS 1 CTD内的调控基序。 工程化的ROS 1 CTD截短物保留催化活性，但在稳态下表现出更长的蛋白质半衰期 和TKI治疗的情况。在目标2中，我们将绘制泛素化位点，跟踪泛素化的细胞命运， ROS 1，并评估体细胞CTD突变对ROS 1功能的影响。 累积起来，我相信这些研究将为ROS 1提供新的机制见解，并可能 促进基因组测序数据的临床翻译，以扩大ROS 1-TKI的影响。

PROJECT SUMMARY Chromosomal rearrangements of the proto-oncogene ROS1 produce constitutively active ROS1 kinase-fusion proteins that are established as druggable pathogenic drivers in human cancer. Currently, this is the only validated mechanism of aberrant ROS1 activation in oncogenesis. ROS1 is targetable with multiple tyrosine kinase inhibitors (TKI), and significant tumor regression is observed in ROS1-fusion positive patients who are treated with targeted TKI. Cancer genome sequencing studies reveal numerous ROS1 somatic mutations but their impact on catalytic function has not been tested, prompting new questions about ROS1-driven cancer pathogenesis. Further, the mechanistic role of ROS1 carboxy terminus in governing intra- or intermolecular regulation is unknown. Broadly, these gaps in knowledge, regarding regulation of ROS1 and its structure-function relationships, impede meaningful utilization of accumulating cancer genome data. Our overall hypothesis is that characterization of ROS1 tyrosine kinase and carboxy-terminal domains through the lens of somatic mutations will unveil biological underpinnings of receptor regulation, and contribute to translation of cancer genomic data. We will experimentally address this hypothesis by answering the following questions: (1) Do cancer-associated ROS1 tyrosine kinase domain (TKD) mutations activate catalytic function, and is this sufficient for neoplastic transformation or metastatic dissemination, either alone or in cooperation with hotspot oncoproteins? Preliminary data offer proof of concept that an engineered activating ROS1 TKD mutation transforms cells, and is targetable with ROS1-TKI. We will test if twenty prioritized somatic ROS1 TKD mutations enhance catalytic function, induce cellular transformation and tumor formation, and assess their drug sensitivity patterns. (2) How does the carboxy-terminal domain (CTD) of ROS1 regulate protein stability and TKI-induced protein downregulation? Our pilot data show that (a) ROS1 is ubiquitinated, (b) catalytically inactive ROS1 undergoes proteasome-assisted degradation, and (c) regulatory motifs within ROS1 CTD are likely involved. Engineered ROS1 CTD truncations retain catalytic activity but exhibit longer protein half-life under steady state and TKI-treated conditions. In Aim 2, we will map ubiquitination sites, track the cellular fate of ubiquitinated ROS1, and assess impact of somatic CTD mutations on ROS1 function. Cumulatively, I am confident that these studies will provide novel mechanistic insight into ROS1, and potentially facilitate clinical translation of genomic sequencing data for expanded impact of ROS1-TKI.