Abstract 2494: Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer (2022)

Abstract Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and young adults. We have shown that all patients have a disruption in the ecology of protein kinase A activity. Hundreds of patients tested have a fusion of the first exon of DNABJ1, a heat shock protein cofactor, to PRKACA, the catalytic subunit of protein kinase A and expression of this DNAJB1-PRKACA fusion oncokinase is sufficient to produce the tumor. One patient is missing the regulatory subunit of protein kinase A and three patients have a fusion of the first exon of a different protein, ATP1B1, to the catalytic subunit of protein kinase A. We characterized the proteome and phosphoproteome of FLC cells relative to adjacent normal tissue. The changes were sufficiently characteristic to identify cell extracts from FLC, based solely on the proteome or phosphoproteome, and distinguishable from other tumor or normal liver. By calibrating protein level we found that tumor cells have an increase of catalytic subunit to such an extent as to exceed the capacity of protein kinase A regulatory subunits. This has two implications. First, there is free catalytic subunit that is unregulated, creating a high basal level of kinase activity. Second, the catalytic subunit is no longer localized by tethering to the regulatory subunit. Thus, the catalytic subunit has access to novel substrates. As an independent confirmation, we found the free basal kinase activity was higher in FLC cells. As a further test, we showed free catalytic subunit, that is not conjugated to regulatory subunit, is higher in FLC cells. We next tested whether these changes were solely due to overexpression of the catalytic subunit or if there was some additional change in the intrinsic activity of the kinase as a result of the fusion. We purified, without using an affinity tag, either PRKACA (which is myristoylated), DNAJB1-PRKACA, and PRKACA which was not myristoylated (the DNAJB1-PRKACA is not myristoylated). As a further control we also used the PRKACAL206R mutation, which does not engage regulatory subunits and is present in some forms of Cushing’s disease. We purified cytosol from human liver, blocked activity of all the endogenous kinases, and added our purified kinase. Mass spectrometry was then used to identify all newly phosphorylated proteins. While many proteins were equally phosphorylated by all four kinases (PRKACA, DNAJB1-PRKACA, PRKACA not myristoylated, PRKACAL206R) there were some distinct differences. From an analysis of these we found alterations in the optimal recognition sequence for phosphorylation for each variant of the kinase. Significantly, when we examined the human tissue data, we found many proteins that were phosphorylated by the DNAJB1-PRKACA in vitro were also phosphorylated in patient tumor tissue, but not in adjacent non-transformed tissue. This validated these observations of altered phosphorylation from the in vitro assays. Citation Format: Solomon Levin, Mahsa Shironi, Melissa Jarmel, Michael Tomasini, Bassem Shebl, Tova Finkelstein, David Requena, Soeren Heissel, Henrik Molina, Philip Coffino, Barbara Lyons, Sanford M. Simon. Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2494.