Decoding the Molecular and Cellular Mechanisms of Mutant KRAS-driven Brain Arteriovenous Malformations

摘要 脑动静脉畸形(BAVM)是由动脉和静脉之间的异常连接组成的 缺乏一个中间的毛细血管网络。因此，来自供血动脉的高压血液直接分流。 变成了血管。这些血管病变变得扩张和高度重塑，导致增大的缠绕 容易破裂的血管。事实上，脑动静脉畸形是导致儿童出血性中风的主要原因。 和年轻人。所有目前的bAVM治疗方式，包括手术、栓塞或放射治疗 有很大的残疾或死亡风险，这些选择不适用于约20%的bAVM患者，原因是 风险过大。由于这些并发症，其他发病率较低的医疗策略，如 人们迫切需要有针对性的药物治疗。然而，我们首先需要清醒地认识到 BAVM发育和维护的生物学基础。大多数bAVM是零星发生的，没有 这种疾病的家族病史。使用整个外显子组测序，我们最近发现了体细胞，激活 KRAS基因突变，该基因编码一种参与信号转导的GTPase。被识别的人 突变仅限于内皮细胞，导致KRAS被锁定在GTP结合的“开”状态。值得注意的是， 我们已经建立了突变的KRAS的内皮特异性表达的小鼠和斑马鱼模型，该突变KRAS 已经揭示了这些基因损伤足以驱动疾病。我们继续向大家展示， 通过培养细胞的转录图谱和表达突变KRAS的斑马鱼体内研究， KRAS诱导的许多分子和细胞变化需要MEK/ERK活性。还有很多东西需要学习 关于散发性bAVM的病因和我们的细胞培养，小鼠和斑马鱼模型将使我们能够 明确参与启动和维持的分子、细胞和形态变化 BAVM。我们将利用我们在bAVM动物模型、成像、细胞生物学、信号和单细胞方面的专业知识。 RNA测序，以获得对bAVM疾病过程的前所未有的洞察力。这一信息将是 用于设计药物干预措施，以改善患者的预后。我们的建议将：1) 定义可重塑血管的KRAS突变内皮细胞阈值，2)识别血管床(S) 3)决定KRAS突变如何影响血流动力学 信号和bAVM进程，4)揭示了细胞自主和非细胞自主机制 突变的KRAS，以及4)确定KRAS和MEK激活在我们的 临床前模型。总之，这些研究将扩大我们对bAVM发病机制的理解，并将 评估抑制KRAS/MEK通路是否可能是人类可行的治疗目标 脑动静脉畸形患者。

SUMMARY Brain arteriovenous malformations (bAVMs) are composed of abnormal connections between arteries and veins that lack an intervening capillary network. As a result, high-pressure blood from feeding arteries shunts directly into veins. These vascular lesions become distended and highly remodeled, resulting in a tangle of enlarged blood vessels that are prone to rupture. Indeed, bAVMs are a leading cause of hemorrhagic stroke in children and young adults. All current treatment modalities for bAVMs, including surgery, embolization or radiation carry a significant risk of disability or death, and these options are not available for ~20% of bAVM patients due to excessive risk. Because of these complications, alternative medical strategies with lower morbidities such as targeted pharmacological therapies are desperately needed. However, we first need a clear understanding of the biology underlying bAVM development and maintenance. The majority of bAVMs occur sporadically without a family history of the disease. Using whole exome sequencing, we recently identified somatic, activating mutations in the KRAS gene, which encodes a GTPase that is involved in signal transduction. The identified mutations were confined to the endothelium and result in KRAS being locked in a GTP-bound ‘ON’ state. Notably, we have established mouse and zebrafish models of endothelial-specific expression of mutant KRAS, which have revealed the sufficiency for these genetic lesions to drive disease. We have gone on to demonstrate, through transcriptional profiling of cultured cells and in vivo studies in zebrafish expressing mutant KRAS, that many KRAS-induced molecular and cellular changes require MEK/ERK activity. Much remains to be learned regarding the etiology of sporadic bAVMs and our cell culture, mouse and zebrafish models will enable us to define the molecular, cellular and morphological changes that are involved in the initiation and maintenance of bAVMs. We will utilize our expertise in animal models of bAVMs, imaging, cell biology, signaling and single-cell RNA sequencing, to gain unprecedented insight into the bAVM disease process. This information will be leveraged for the design of pharmacological interventions to improve patient outcomes. Our proposal will: 1) define the threshold of KRAS mutant endothelial cells that can remodel vessels, 2) identify the vascular bed(s) that are susceptible to active KRAS expression, 3) determine how KRAS mutations impact hemodynamic signaling and bAVM progression, 4) uncover the cell-autonomous and non-cell autonomous mechanisms of mutant KRAS, and 4) determine the requirement for KRAS and MEK activation for bAVM maintenance in our pre-clinical models. Together, these studies will expand our understanding of bAVM pathogenesis and will assess whether inhibition of the KRAS/MEK pathway may be a viable therapeutic target to pursue in human patients with bAVM.