Abstract B034: Mechanisms underlying Fibrolamellar Carcinoma

Introduction: Many pediatric cancers, are driven by fusion oncoproteins. Many of the oncogenic fusions are to kinases such as ALK, ROS1, RET, NTRK3, FGFR3, MET, ABL1, JAK2, and BRAF. The mechanism for these are believe to be the consequence of altered function such as constitutive dimerization, loss of autoinhibition or aberrant cellular localization. Fibrolamellar carcinoma (FLC) is driven by the fusion of the first exon of DNAJB1, a heat shock protein, to the bulk of the coding region of PRKACA, the catalytic subunit of protein kinase A (PKA), forming DNAJB1::PRKACA. The active sites of the DNAJB1::PRKACA and native PRKACA are indistinguishable structurally and enzymatically raising the question of how does formation of the DNAJB1::PRKACA lead to transformation. Brief summary: We examined the possibility that increased activity levels of protein Kinase A induces FLC, independent of the fusion adduct, DNAJB. In FLC, expression of DNAJB1::PRKACA fusion is driven by the heat shock promoter. PKA is a holoenzyme of two catalytic subunits and two regulatory subunits, who inhibit and localize the catalytic activity. Results: Quantitative mass spectrometry demonstrated that in patient normal liver there is a quantitative excess of regulatory subunit but in FLC tumor tissue there is an excess of catalytic subunit. Further, many of the regulatory subunits in condensates which are low, or missing catalytic subunit. The net result is a large increase of catalytic activity and a large increase of catalytic subunit in the nucleus. Overexpression of either the native PRKACA or the fusion DNAJB1::PRKACA in primary human hepatocytes produced indistinguishable alterations of the transcriptome, and they both recapitulated the transcriptome in patient tumors. We found patients who had fusions of a different domain to PRKACA, which also resulted in overexpression of the catalytic subunit, and their transcriptomes were the same as in FLC and their drug response properties were the same. We found patients with no fusion to the catalytic subunit but they were missing the regulatory subunit and their transcriptome and histopathology was indistinguishable from DNAJB1::PRKACA driven FLC. Conclusion: FLC is driven by the overexpression of the catalytic activity of protein kinase A, not by an altered or gain of function. Citation Format: Mahsa Shirani, David Requena, Denise Ng, Philip Coffino, Barbara Lyons, Sanford M. Simon. Mechanisms underlying Fibrolamellar Carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B034.