A novel high-throughput screening platform to identify inhibitors of DNAJB1-PRKACA-driven transcriptional activity in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a primary liver cancer with a poor prognosis, primarily due to the lack of effective chemotherapeutic options. The DNAJB1-PRKACA (DP) gene fusion is recognized as the key oncogenic driver in FLC. This fusion arises from a ∼400 kb heterozygous deletion on chromosome 19, which fuses exon 1 of DNAJB1 with exons 2-10 of PRKACA, the gene encoding the catalytic subunit of protein kinase A (PKA). While targeting DP is considered a promising therapeutic approach, attempts to inhibit the kinase function of the DP fusion protein have been largely unsuccessful due to off-target effects on wild-type PKA. In response to this challenge, we developed a high-throughput screening (HTS) assay to identify inhibitors of DP's downstream signaling pathways involved in transcriptional regulation. Our previous research identified LINC00473 as a transcriptional marker for DP protein expression, and LINC00473 is known to be upregulated in FLC tumors. Additionally, evidence suggests that LINC00473 promotes FLC tumor growth. Based on the relationship between DP and LINC00473 expression, we engineered the HEK-DP-Luc reporter cell line by modifying HEK293 cells to express DP at the endogenous locus and to express the NanoLuc luciferase gene under the control of the LINC00473 promoter and enhancer. We have optimized the HEK-DP-Luc cells for HTS, and here we present our pipeline for primary screening and counter-screening to identify compounds that inhibit DP's downstream transcriptional activity. This HTS platform provides a novel approach for therapeutic drug discovery in FLC.