Biology of R-Spondin-Induced Sensitization to Asparaginase in Colorectal Cancer

摘要 结直肠癌（CRC）是美国癌症死亡的第二大原因，与许多其他肿瘤不同， 类型，没有已知的有效疗法靶向显性致癌驱动因子。几乎所有的CRC 激活经典Wnt/β-catenin信号传导的突变，但直接抑制β-catenin是困难的， 阻断Wnt配体活性导致显著的靶向骨毒性。因此，虽然直接靶向Wnt是 具有挑战性的异常Wnt通路激活可能会诱导肿瘤特异性脆弱性， CRC治疗。使用全基因组遗传筛选，我们发现Wnt激活诱导了深刻的 在耐药白血病中使用天冬酰胺酶对治疗性天冬酰胺消耗的敏感性。这种效果是 依赖于Wnt诱导的GSK 3抑制，但不依赖于APC或β-连环蛋白。相反，天冬酰胺酶 致敏作用是由Wnt诱导的对GSK 3依赖性蛋白降解的抑制介导的，GSK 3依赖性蛋白降解是一种分解代谢源， 天冬酰胺酶抗性所需的氨基酸。CRC提供了一个独特的测试环境 因为这些肿瘤几乎都有激活Wnt/β-catenin的突变， 这些可以在GSK 3的上游或下游起作用。使用人CRC细胞系和遗传学方法， 我们发现，天冬酰胺酶对具有以下突变的CRC几乎没有影响： 下游Wnt因子APC，但对R-spondin易位的病例具有深刻的毒性， 通过配体诱导的GSK 3抑制激活Wnt信号传导，从而抑制GSK 3依赖性蛋白 降解重要的是，这种方法对正常肠或上皮细胞几乎没有可检测的毒性。这 表明这种方法具有有效的治疗指数，可以改变患者的临床结果。 每年有数千名患者死于CRC，基于这些数据的临床试验正在进行中。 发展然而，我们不了解这种RSPO/Wnt诱导的生物学的关键方面。 治疗脆弱性。确定决定RSPO/Wnt诱导的天冬酰胺酶的精确分子事件 灵敏度对于前瞻性识别临床应答者、设计合理的方法以改善 治疗反应和克服治疗抗性。这些知识差距将在 以下目的：1）确定RSPO配体如何诱导对天冬酰胺酶的敏化。2)调查的作用 GSK 3 α体的形成是对天冬酰胺饥饿的细胞反应。3)确定致癌的作用 RSPO融合CRC对天冬酰胺酶的治疗反应中的KRAS和TP 53突变。这项建议是 预计将提供基本的见解氨基酸饥饿反应及其损害， 异常Wnt信号传导，是后生动物生命的基本细胞过程，其分子基础和 对治疗性剥削仍然知之甚少。鉴于我们在天冬酰胺酶方面的专业知识高度互补 生物学和Wnt信号，这个MPI团队是独一无二的准备将这些进展转化为高度创新的 治疗干预。

ABSTRACT Colorectal cancer (CRC) is the second-leading cause of cancer deaths in the US, and unlike many other tumor types, there are no known effective therapies targeting dominant oncogenic drivers. Almost all CRCs have mutations that activate canonical Wnt/β-catenin signaling, but direct inhibition of β-catenin is difficult, and blocking Wnt ligand activity leads to significant on-target bone toxicity. Thus, while targeting Wnt directly is challenging, aberrant Wnt pathway activation may induce tumor-specific vulnerabilities that can be exploited for CRC therapy. Using a genome-wide genetic screen, we found that Wnt activation induces profound sensitization to therapeutic asparagine depletion using asparaginase in drug-resistant leukemias. This effect is dependent on Wnt-induced inhibition of GSK3, but is independent of APC or β-catenin. Instead, asparaginase sensitization is mediated by Wnt-induced inhibition of GSK3-dependent protein degradation, a catabolic source of amino acids required for asparaginase resistance. CRC provides a unique context in which to test predictions from our model, because these tumors almost all have mutations that activate Wnt/β-catenin, but these can function either upstream or downstream of GSK3. Using human CRC cell lines and genetically engineered mouse intestinal organoids, we found that asparaginase had little effect on CRCs with mutations of the downstream Wnt factor APC, but was profoundly toxic to cases with R-spondin translocations, which activate Wnt signaling via ligand-induced inhibition of GSK3, and thus inhibit GSK3-dependent protein degradation. Importantly, this approach has little detectable toxicity to normal intestinal or epithelial cells. This suggests that this approach has a potent therapeutic index that could transform clinical outcomes for the thousands of patients who die of CRC every year, and a clinical trial based on these data is under development. However, we do not understand key aspects of the biology underlying this RSPO/Wnt-induced therapeutic vulnerability. Defining the precise molecular events that dictate RSPO/Wnt induced asparaginase sensitivity is critical for prospectively identifying clinical responders, designing rational approaches to improve therapeutic response, and overcoming treatment resistance. These knowledge gaps will be addressed in the following Aims: 1) Determine how RSPO ligands induce sensitization to asparaginase. 2) Investigate the role of GSK3α body formation as a cellular response to asparagine starvation. 3) Determine the role of oncogenic KRAS and TP53 mutations in therapeutic response of RSPO fusion CRC to asparaginase. This proposal is expected to provide fundamental insights into the amino acid starvation response and its impairment by aberrant Wnt signaling, cellular processes fundamental to metazoan life whose molecular basis and therapeutic exploitation remain poorly understood. Given our highly complementary expertise in asparaginase biology and Wnt signaling, this MPI team is uniquely poised to translate these advances into highly innovative therapeutic interventions.