Author ORCID Identifier
https://orcid.org/0000-0002-3347-3045
Date of Graduation
12-2023
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Lawrence Kwong
Committee Member
Jian Hu
Committee Member
Traver Hart
Committee Member
Min Gyu Lee
Committee Member
Haoqiang Ying
Abstract
Biliary tract cancer (BTC) is malignant tumor that originates from the biliary tract, including cholangiocarcinoma (CCA) and gallbladder cancer (GBC). In this translational research program targeting BTC, we focus on three aspects: the development of a combination treatment against CCA, the exploration of the immune background of GBC, and the establishment of a CCA mouse model mimicking mutation profiles of CCA patients.
Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify novel drug combinations for this disease, we have conducted a genome wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the known regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tied together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.
Gallbladder carcinoma is a deadly disease with a poor prognosis and improving immunotherapeutic approaches for this disease will require a detailed understanding of its immunogenomic profile. In this study, we combined targeted next generation sequencing and immunohistochemistry to analyze 122 gallbladder carcinoma tumor tissue samples from two cohorts in the United States (n=60) and Chile (n=62). We identified distinct immune profiles, as the Chilean primary cohort had higher densities of biomarkers associated with T cells and immune checkpoints, while the USA primary cohort showed higher densities of tumor-associated macrophages. The samples were also categorized into four immune profile subtypes using unsupervised clustering. Finally, we identified preliminary associations between certain immune markers and CCNE1 amplifications, gender, or tumor stage. Our results provide a foundation for larger scale immunogenomic studies of gallbladder carcinoma and may aid in the development of improved immunotherapies for this disease.
To deepen our understanding of CCA, we developed a novel mouse model of CCA which harbors genetic mutation combinations matching CCA patient mutation profiles, with no hepatocellular carcinoma development, accompanied by an intact immune tumor microenvironment. To achieve this goal, we used hydrodynamic tail vein injection (HTVI) method, in which oncogenes and CRISPRs targeting tumor suppressor genes are encoded in Sleeping Beauty (SB) transposon plasmids, which are permanently delivered via integration into liver cell genomes. Mice were also treated with the liver injury agent Diethyldithiocarbamate (DDC) for 4 weeks to expedite the development of CCA. To identify the pathological characteristics of mice tumor, H&E staining, and Immunohistochemistry (IHC) targeting oncogenes and HCC/CCA markers were applied. We successfully developed two CCA models, one is Kras-G12D/sgPten/NICD1 and another is FGFR2-BICC1/sgTP53/NICD1. Based on these mice models, intratumor heterogeneity and diverse drug responses will be explored. To improve the ratio of well differentiated CCA, we will use bile duct ligation technique instead of HTVI to make sure that plasmids can enter biliary tract directly.
In summary, we found a novel combination of BET and mTOR inhibitors to against CCA, which paves a way for future clinical trials based on this combination; explored a deeper and more comprehensive immunogenomic profile of GBC, which provides a rationale for the application of multiple immune checkpoint inhibitors against GBC; and established a CCA mouse model representing CCA patient mutation profiles, which can be used to explore intratumor heterogeneity and diverse drug responses.
Keywords
Biliary tract cancer, gallbladder cancer, cholangiocarcinoma