RNA Therapeutics for Fibrolamellar Hepatocellular Carcinoma

Author

Christoph Neumayer

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer characterized by a recurrent fusion of the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A (PRKACA). Due to limited efficacy of conventional treatments such as chemotherapy and radiation my work explored new therapeutic options. As DNAJB1::PRKACA is known to initiate tumorigenesis, I reasoned that it would be an ideal target for a therapeutic. However, it was not known if FLC tumors are still dependent on DNAJB1::PRKACA or if cells achieved independence of the initial oncogene. To test if FLC is dependent on the oncokinase I screened short hairpin RNAs (shRNAs) that tile over the fusion junction of the DNAJB1::PRKACA mRNA. I identified a shRNA that achieves strong knockdown of the fusion gene with minimal effect on the wildtype fusion partners.Using this shRNA, I demonstrate that specifically knocking down DNAJB1::PRKACA results in the cell death of FLC cells in vitro and in patient derived xenograft models of FLC in mice. These results show that DNAJB1::PRKACA has a pivotal role in tumor formation, maintenance, and progression and that FLC is oncogenically dependent on DNAJB1::PRKACA. This validates DNAJB1::PRKACA as a therapeutic target. To translate this finding into a potential therapeutic, I collaborated with Ionis Pharmaceuticals to synthesize small interfering RNAs (siRNAs) with state-of-the-art chemical modifications. Multiple siRNA therapeutics targeting genes in the liver have been FDA approved. The majority of these use a carbohydrate group, GalNAc, that binds to the asialoglycoprotein receptor (ASGR), allowing targeted delivery to hepatocytes. I hypothesized that ASGR might still be sufficiently expressed in FLC tumors to deliver siRNAs to cells in vivo. I demonstrated that indeed ASGR is still expressed across different stages of the FLC in patient samples and PDX models. Using PDX cells of patients, I show that GalNAc-conjugated siRNAs can be internalized via ASGR in vitro and result in knockdown of DNAJB1::PRKACA and cell death. In two different FLC PDX lines of subcutaneous and splenic tumor models, siRNAs targeting DNAJB1::PRKACA are effectively taken up in vivo with strong and durable target knockdown and tumor growth inhibition. To further enhance the delivery of RNA therapeutics to FLC cells, I developed lipid nanoparticles(LNPs) based on the ionizable lipid C12-200. These LNPs effectively delivered mRNA encoding luciferase to cells in vitro, demonstrating their potential for delivering therapeutic RNA payloads. However, delivery to FLC cells in vivo remains challenging. Additionally, I performed cell surface proteomics using a proximity-based labeling approach to identify potential targets for antibody-based targeting strategies. Comparative analysis revealed distinct surface protein profiles between FLC and hepatocellular carcinoma (HCC) spheroids. Validation of selected targets in patient samples confirmed their differential expression and localization in FLC tumors compared to healthy liver tissue, providing a valuable resource for developing targeted therapies for FLC. The successful knockdown of this fusion gene through inducible shRNA and siRNA strategies offers a promising treatment avenue for FLC patients, underscoring the potential of mRNA degrading modalities such as antisense oligonucleotides, siRNAs, and possibly PROTACS for targeted intervention. This work establishes a foundation for the development of precision therapies against FLC, highlighting the importance of the DNAJB1::PRKACA fusion kinase as a therapeutic target and serving as a model for targeting other fusion genes in pediatric and possibly adult cancers, paving the way for more effective treatments.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Neumayer, Christoph, "RNA Therapeutics for Fibrolamellar Hepatocellular Carcinoma" (2024). Student Theses and Dissertations. 769.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/769