Epigenetic mechanisms of transcriptional activation of a novel oncogenic ALK variant in cancer

摘要 癌症的全面基因组表征彻底改变了我们对癌症的理解 发病机制，并为靶向治疗在各种疾病中的临床成功提供了科学依据。 癌的有新的证据表明，通过表观遗传机制激活的癌基因也是 在致癌转化中很重要，并且可以决定治疗反应。我们最近发现了一本小说 致癌ALK变体ALKATI，通过不依赖于遗传的交替转录起始产生 在所有TCGA癌症的3.4%中，包括12%的黑色素瘤，在其他癌症类型中频率较低。 ALKATI可以刺激多种信号传导途径，并能够驱动肿瘤细胞的致癌转化。 在体外使细胞永生化并在体内驱动肿瘤发生。此外，工程改造的ALKATI转化细胞和 肿瘤和ALKATI阳性患者来源的细胞系和异种移植物对ALK抑制剂敏感。患者 患有ALKATI阳性晚期黑色素瘤，在标准治疗全身治疗中进展， 纳武单抗和伊匹单抗研究性联合免疫疗法获得显著的临床益处 克唑替尼（一种ALK抑制剂）治疗，肿瘤缩小，生活质量改善。这些数据 为当前ALK靶向治疗的临床研究提供了科学依据和热情 治疗难治性晚期黑色素瘤 ALKATI在正常组织中不表达。它在肿瘤样品中双等位基因表达。这些观察， 以及ALKATI阳性肿瘤样本中ALK基因座缺乏体细胞突变，表明 ALKATI转录激活是通过表观遗传机制。我们的初步数据进一步表明 MAPK信号调节ALKATI表达。在此，我们提出系统全面的 研究集中在以下三个目标的理解表观遗传机制：1）定义 与ALKATI转录因子的长程相互作用元件的三维染色质组织 起始位点（ALK-ATI位点）; 2）鉴定和表征参与ALKATI的染色质修饰剂 转录调控; 3）阐明ALKATI表达通过MAPK信号转导的调节机制， 黑素瘤我们将使用现代技术，包括RNA-seq，ChIP-seq，4C-seq，CRISPR，CRISPR 干扰（CRISPRi）系统，生物信息学和综合分析，以及一个良好表征的 用于拟定多学科研究的ALKATI阳性和ALKATI阴性细胞系。我们预计 这些机制的研究不仅将提供对癌基因的基本表观遗传机制的了解， 癌症中通过交替转录起始的转录激活，以及新的表观遗传 通过类似机制靶向癌基因转录激活的治疗策略。

Abstract Comprehensive genomic characterization in cancer has revolutionized our understanding of cancer pathogenesis and provided the scientific rationale for the clinical success of targeted therapies in a variety of cancers. There is emerging evidence that oncogenes activated through epigenetic mechanisms are also important in oncogenic transformation and can dictate therapeutic responses. We recently discovered a novel oncogenic ALK variant, ALKATI, arising through alternative transcription initiation independent of genetic alteration, in 3.4% of all TCGA cancers, including 12% of melanomas and less frequently in other cancer types. The ALKATI can stimulate multiple signaling pathways and is capable of driving oncogenic transformation in immortalized cells in vitro and drive tumorigenesis in vivo. Moreover, engineered ALKATI-transformed cells and tumors, and ALKATI-positive patient derived cell lines and xenografts are sensitive to ALK inhibitors. A patient with ALKATI-positive advanced melanoma who has progressed on standard of care systemic therapy and investigational combination immunotherapy of nivolumab and ipilimumab derived significant clinical benefit from Crizotinib (an ALK inhibitor) with tumor shrinkage and improvement in quality of life. These data have provided the scientific rationale and enthusiasm for the current clinical investigations of ALK-targeted therapies in therapeutic refractory advanced melanoma. ALKATI is not expressed in normal tissues. It is biallelically expressed in tumor samples. These observations, together with the lack of somatic mutations at the ALK locus in the ALKATI-positive tumor samples, indicate that the ALKATI transcriptional activation is through epigenetic mechanisms. Our preliminary data further indicate that MAPK signaling regulates ALKATI expression. Here, we proposes systematic and comprehensive investigations focused on understanding the epigenetic mechanisms in the following three aims: 1) Define the three-dimensional chromatin organization of the long-range interacting elements with the ALKATI transcriptional start site (the ALK-ATI site); 2) Identify and characterize the chromatin modifier(s) involved in ALKATI transcriptional regulation; 3) Elucidate the regulatory mechanisms of ALKATI expression by MAPK signaling in melanoma. We will use modern technologies, including RNA-seq, ChIP-seq, 4C-seq, CRISPR, CRISPR interference (CRISPRi) systems, bioinformatics and integrative analyses, and a repertoire of well-characterized ALKATI-positive and ALKATI-negative cell lines for the proposed multidisciplinary studies. We anticipate that these mechanistic studies will provide insight on not only the basic epigenetic mechanisms in oncogene transcriptional activation through alternative transcription initiation in cancer, but also novel epigenetic therapeutic strategies to target the oncogene transcriptional activation through similar mechanisms.