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.