Dissecting the Mechanism of SETDB1 and its K867 Monoubiquitination in Lung Cancer Progression

项目摘要 SETDB1及其K867单素化在肺癌进展中的作用机制剖析 非小细胞肺癌(NSCLC)是全球癌症相关死亡的主要原因之一。 并与高突变负担相关。在过去的十年中，生物标记物驱动的靶向治疗已经 在具有多种致癌驱动基因突变的患者中获得了明显的临床益处，如EGFR、BRAF和 好的。然而，具有最普遍的KRAS突变的肿瘤的治疗仍然具有挑战性，尽管 最近在靶向KRAS-G12C突变方面的突破。在KRAS突变体NSCLC中，成分激活的 下游信号最终导致转录重新编程，以维持肿瘤的生长和进展。 这一过程是由一组关键的转录因子编排的，它们的时间和空间表达 与明显的表观遗传学变化有关。因此，剖析这一过程中的表观遗传机制可能 为目标策略开辟新的途径。SETDB1是催化组蛋白H3K9的主要甲基转移酶 甲基化，一种主要的抑制性表观遗传修饰。SETDB1基因的焦点扩增和上调 已在包括非小细胞肺癌在内的多种癌症中观察到，表明它具有促肿瘤作用。 然而，SETDB1在非小细胞肺癌中的作用机制仍不清楚。在初步研究中，我们发现 肺腺癌(ADC)中SETDB1表达上调与预后不良显著相关 与KRAS基因交替共发生。在含有致癌RAS的NSCLC细胞中，SETDB1丢失减弱 迁移和入侵、内生足纲的形成和细胞外基质的降解。这些表型变化与 转录因子FOXA2和CDX2的重新激活，这两个转录因子作为关键调节节点来阻碍 KRAS突变型肺ADC的转移进展。更重要的是，肿瘤细胞中的SetDB1丢失源于 转移性KrasG12D；p53f1/fl肺ADC丧失了皮下转移的自发能力 肺部有肿瘤。这些发现支持一个新的概念，即SETDB1介导的表观遗传机制 在肺ADC进展过程中，对致癌的KRAS诱导的转录重编程至关重要。此外， 我们已经证明了SETDB1在赖氨酸-867处被UBE2E家族的E2酶单核苷酸修饰 不依赖于E3S，这种单泛素化对于SETDB1介导的H3K9甲基化和ERV是必不可少的 沉默。因此，这项建议的一个目标是描绘SETDB1‘S在KRAS中的促转移活性 使用各种基因工程小鼠模型和异种移植模型的突变肺ADC。另一个目标 旨在探讨赖氨酸-867单素化在SETDB1‘S体内促转移活性中的作用机制。 在试管中。我们的中心假设是SETDB1在KRAS突变的肺ADC进展中起重要作用 通过表观遗传机制，需要其依赖于单泛素化的酶活性。这个项目 意义重大，因为它将把SETDB1表征为KRAS突变肺癌的可操作药物靶点 并确定用于靶向的新的分子界面。我们的长远目标是发展得更好、更有成效 通过描绘肺癌的表观遗传机制的治疗策略。

Project Summary Dissecting the Mechanism of SETDB1 and its K867 Monoubiquitination in Lung Cancer Progression Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality worldwide and associated with high mutation burdens. During the last decade, biomarker-driven targeted therapies have achieved clear clinical benefits in patients with several oncogenic driver mutations such as EGFR, BRAF and ALK. However, the treatments for tumors with the most prevalent KRAS mutations remain challenging despite of recent breakthrough in targeting KRAS-G12C mutation. In KRAS mutant NSCLC, the constitutively activated downstream signaling ultimately leads to transcription reprogramming to sustain tumor growth and progression. This process is orchestrated by a cohort of crucial transcription factors, whose temporal and spatial expression is associated with distinct epigenetic changes. Thus, dissecting epigenetic mechanisms in this process could open new avenues for targeting strategies. SETDB1 is a principal methyltransferase catalyzing histone H3K9 methylation, a major repressive epigenetic modification. The focal amplification and upregulation of SETDB1 have been observed in a wide range of cancers including NSCLC, suggesting it has pro-oncogenic function. However, the mechanisms of action of SETDB1 in NSCLC remain elusive. In preliminary study, we uncovered that SETDB1 upregulation in lung adenocarcinoma (ADC) correlates with poorer prognosis and significantly co-occurs with KRAS gene alternations. In NSCLC cells harboring oncogenic RAS, SETDB1 loss attenuates migration and invasion, invadopodia formation and ECM degradation. These phenotypic changes correlate with reactivation of transcription factors FOXA2 and CDX2 which function as key regulatory nodes to impede metastatic progression of KRAS mutant lung ADC. More importantly, Setdb1 loss in tumor cells derived from metastatic KrasG12D;p53fl/fl lung ADC abolished their spontaneous ability to metastasize from the subcutaneous tumor to the lungs. These findings argue for a novel concept that SETDB1-mediated epigenetic mechanisms are critical for oncogenic KRAS-induced transcription reprogramming during lung ADC progression. Moreover, we have demonstrated that SETDB1 is monoubiquitinated at lysine-867 by the UBE2E family of E2 enzymes independent of E3s and this monoubiquitination is essential for SETDB1-mediated H3K9 methylation and ERV silencing. Accordingly, one objective of this proposal is to delineate SETDB1's pro-metastatic activity in KRAS mutant lung ADC using various genetically engineered mouse models and xenograft models. Another objective is to dissect the mechanisms of lysine-867 monoubiquitination in SETDB1's pro-metastatic activity in vivo and in vitro. Our central hypothesis is that SETDB1 plays important roles in KRAS mutant lung ADC progression through epigenetic mechanisms that require its monoubiquitination dependent enzymatic activity. This project is significant because it will characterize SETDB1 as an actionable drug target in KRAS mutant lung cancer and identify novel molecular interfaces for targeting. Our long-term goal is to develop better and more effective therapeutic strategies by delineating epigenetic mechanisms in lung cancer.