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

项目总结 原癌基因ROS1的染色体重排产生结构性活性的ROS1激酶融合 在人类癌症中被确定为可药物致病驱动因素的蛋白质。目前，这是唯一的 证实了肿瘤发生中ROS1异常激活的机制。多个酪氨酸靶向的ROS1 激酶抑制物(TKI)，在ROS1融合阳性的患者中观察到显著的肿瘤消退。 靶向TKI治疗。癌症基因组测序研究显示许多ROS1体细胞突变，但 它们对催化功能的影响还没有得到测试，这引发了关于ROS1驱动的癌症的新问题 发病机制。此外，ROS1羧基末端在控制或 分子间的调节尚不清楚。总的来说，关于ROS1监管的这些知识差距 及其结构-功能关系，阻碍了对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ROS1酪氨酸激酶和羧基末端结构域的特征是通过 体细胞突变的镜头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一假设 问题： (1)癌症相关的ROS1酪氨酸激酶域(TKD)突变是否激活了催化功能，以及 无论是单独还是合作，这对肿瘤转化或转移扩散来说足够吗？ 用热点癌蛋白？初步数据提供了工程激活ROS1 TKD的概念证据 突变可以转化细胞，并且可以通过ROS1-TKI靶向。我们将测试20个优先的躯体ROS1 TKD 突变增强催化功能，诱导细胞转化和肿瘤形成，并评估其药物 敏感度模式。 (2)ROS1的羧基末端结构域(CTD)是如何调节蛋白质稳定性和TKI诱导的 蛋白质下调？我们的实验数据表明(A)ROS1是泛素化的，(B)催化失活的ROS1 经历蛋白酶体辅助的降解，以及(C)ROS1 CTD内的调节基序可能参与其中。 工程ROS1 CTD截断保留了催化活性，但在稳定状态下显示出更长的蛋白质半衰期 和TKI治疗条件。在目标2中，我们将绘制泛素化位点图，跟踪泛素化的细胞命运 ROS1，并评估体细胞CTD突变对ROS1功能的影响。 总体而言，我相信这些研究将为ROS1提供新的机制洞察力，并有可能 促进基因组测序数据的临床翻译，以扩大ROS1-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.