Structural Validation of a Protein Binder Targeting an Oncogenic Fusion Protein in Fibrolamellar Carcinoma

Abstract

Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of liver cancer that primarily affects children and young adults without the presence of liver disorder. The disease results in poor patient prognosis with only a 45% to 70% survival rate at five years. In 2014, the underlying cause was identified to be the protein product of a gene fusion between the DNAJB1 and PRKACA. The genetic aberration results in a stable fusion protein comprising part of DNAJ, a protein chaperone, and the catalytic subunit of Protein Kinase A (PKAc), which plays many critical roles in cell metabolism and proliferation. While the underlying cause has been replicated in mice, the mechanism by which the aberrant protein drives cancer is still poorly understood. The DNAJ component of the oncogenic fusion protein has been implicated in abnormal protein stability and cellular localization, whereas the PKAc constituent has been speculated to trigger aberrant cell signaling due to mis-localization and uncoupling with its native regulatory subunits. Importantly, many attempts using new and existing drugs to treat FLC have encountered clinical challenges due to ineffectiveness or intolerable side effects. This highlights the unmet medical need for novel therapeutic approaches to treating this disease. This study investigates the structural basis of a designed protein binder that selectively targets the unique fusion junction of DnaJ-PKAc as a modular tool for developing novel therapeutic strategies for FLC. Recombinant expression and purification of DnaJ-PKAc, KL_D1, and the RIIβ regulatory subunit of PKA were successfully established. The binary DnaJ-PKAc:KL_D1 complex were reconstituted and confirmed by size exclusion chromatography and SDS-PAGE. Crystallization screening across multiple commercial sparse-matrix screens yielded several crystal candidates. However, crystal fragility, small crystal size, and the inherent conformational dynamics of the apo PKAc subunit limited the diffraction power of these crystals. Further optimization of the crystallization conditions and alternative structural approaches will help gain atomic-resolution insight into the DnaJ-PKAc:KL_D1 binding interface.