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

摘要 癌症的全面基因组特征彻底改变了我们对癌症的理解 并为靶向治疗在多种肿瘤中的临床成功提供了科学理论基础。 癌症。有新的证据表明，通过表观遗传机制激活的癌基因也 在致癌转化中很重要，可以决定治疗反应。我们最近发现了一本小说 癌基因ALK变异体，ALKATI，通过不依赖于遗传的选择性转录启动而产生 改变，在所有TCGA癌中，3.4%，包括12%的黑色素瘤，在其他类型的癌症中较少发生。 ALKATI可以刺激多条信号通路，并能够推动肿瘤的转化 在体外使细胞永生，并在体内推动肿瘤的形成。此外，工程化的ALKATI转化细胞和 肿瘤、ALKATI阳性患者来源的细胞系和异种移植对ALK抑制剂敏感。一位病人 ALKATI阳性的晚期黑色素瘤患者在标准护理和系统治疗方面取得了进展 尼伏单抗和伊普利单抗联合免疫治疗的研究取得了显著的临床益处 来自Crizotinib(一种碱性磷酸酶抑制剂)，具有肿瘤缩小和生活质量改善的作用。这些数据有 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。它在肿瘤样本中以双等位基因表达。这些观察结果， 再加上ALKATI阳性肿瘤样本中ALK基因座缺乏体细胞突变，表明 ALKATI的转录激活是通过表观遗传机制实现的。我们的初步数据进一步表明 MAPK信号调节ALKATI的表达。在这里，我们提出了系统的、全面的 研究集中在以下三个目标上理解表观遗传机制：1)定义 与ALKATI转录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)鉴定与ALKATI有关的染色质修饰物(S) 3)阐明MAPK信号通路对ALKATI基因表达的调控机制。 黑色素瘤。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，以及具有良好特性的曲目 为拟议的多学科研究提供ALKATI阳性和ALKATI阴性的细胞系。我们预料到 这些机制研究将不仅对癌基因的基本表观遗传机制提供洞察力 癌症中通过交替转录启动的转录激活，但也有新的表观遗传学 通过类似的机制针对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.