Molecular interactions and signaling mechanisms of the oncogenic fusion protein DNAJ-PKAc in fibrolamellar carcinoma

Abstract

The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperone binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ-PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and live-cell photoactivation microscopy we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the co-chaperone Bcl-2 associated athanogene 2 (BAG2), a regulator of Hsp70-mediated protein refolding and inhibitor of CHIP-mediated ubiquitination. Immunoprecipitation experiments in AML12 hepatocytes and FLC patient tissue showed co-precipitation of BAG2 with DNAJ-PKAc but not wild-type PKAc. Use of a kinase-dead mutant (K72H) in AML12 cells demonstrated that this interaction is independent of fusion kinase activity and reconstitution of FLC complexes using the SpyCatcher/SpyTag heterodimerization system also confirmed this interaction. Furthermore, immunoblot and immunohistochemical analyses of FLC patient samples demonstrate increased levels of BAG2 in primary tumors and metastatic recurrence. BAG2 is linked to the anti-apoptotic factor Bcl-2. Pharmacological approaches using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax show that the DNAJ-PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in a cellular model of FLC. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.