Fibrolamellar carcinoma (FLC) is a rare form of liver cancer that disproportionately affects adolescents, usually presents at advanced stages with no known risk factors or serum biomarkers, and exhibits high intrinsic drug resistance. Currently, no established therapies exist for this devastating disease. The signature genetic event of FLC was identified in 2014 as a ~400kb somatic, heterozygous deletion resulting in the DNAJB1-PRKACA (or DP) fusion gene. The DP fusion occurs in over 80% of FLC patients and is sufficient for tumorigenesis in mice. Targeting DP pharmacologically has proved challenging; therefore, it is critical to identify and target candidate downstream effectors that drive FLC development and progression. To address this gap in the field, the Sethupathy lab performed multiple genome-scale studies which identified LINC00473, a primate-specific long non-coding RNA (lncRNA), to be dramatically elevated in FLC tumors and associated with a FLC-specific super enhancer region. These findings suggested that LINC00473 may be an important oncogene in FLC. The preliminary data (see next section) strongly motivate my hypothesis that LINC00473 functions as a critical regulator of FLC tumor growth.

Aim 1: Evaluate the effect of LINC00473 knockdown and overexpression in FLC cells in vivo. Emerging evidence demonstrates that lncRNAs are abnormally expressed in tumors and can function as oncogenes in several different cancer types. My preliminary data show that LINC00473 is more aberrantly elevated in FLC than any other tumor type, associated with an FLC-specific super enhancer, responsive to DP fusion activity in several human FLC models, and regulates FLC cell growth in two independent cell models. However, it remains unknown whether LINC00473 regulates FLC tumor growth in vivo. Approach: By conducting gain- and loss-of-function experiments, I will interrogate the effect of LINC00473 on tumor growth via subcutaneous transplantation studies.

Aim 2: Define the cell type-specific expression pattern of LINC00473 in primary FLC tumors. The specific biological roles of lncRNAs depend on the cell type in which they are expressed. FLC tumor tissue comprises many distinct cell types with different functions. However, the expression pattern of LINC00473 across these cell types remains completely unknown. Approach: To address this knowledge gap, I will perform single nucleus RNA-seq on primary FLC tumors to identify the specific cell type populations that express LINC00473, as well as the aberrant cancer signaling pathways associated with LINC00473 expression.

Collectively, this proposal outlines a comprehensive approach that dissects the function of one of the most highly dysregulated, yet poorly understood, genes in FLC. This study will not only provide important insight into the molecular etiology of FLC, thereby potentially opening up novel therapeutic avenues, but also will lay the foundation for the functional characterization of LINC00473 that will advance the compendium of lncRNAs.