Therapeutic Targeting Of Chemotherapy Resistant Colorectal Cancer Stem Cells With P73 And Trail Based Small Molecules PDF Download

The majority of colorectal cancers arise from dysregulation in the Wnt signaling pathway. Aberrant Wnt signaling is associated with tumor formation, metastasis, and drug resistance. Many current therapeutic strategies attempt to target downstream effectors of the Wnt pathway, most notably protein-protein interactions that mediate formation of b-catenin/TCF4 complexes. These strategies have had limited clinical success, due in part to there being a limited amount of known druggable targets involved with activating transcription of Wnt target genes. In an attempt to develop more effective therapies for colorectal cancer, this manuscript describes our efforts to validate CHD1L as a novel druggable target involved in Wnt signaling and identify the first known small molecule inhibitors of CHD1L. CHD1L is an oncogene involved in drug resistance, anti-apoptotic mechanisms, tumor progression, and metastasis in a variety of solid tumors. Prior to the work presented in this dissertation, little was known about the mechanistic role of CHD1L in colorectal cancer and no direct link had been established between CHD1L and Wnt signaling. To validate CHD1L as a potential drug target in the Wnt pathway, we examined gene expression data from colorectal cancer patients and performed in vitro experiments to determine if CHD1L was involved in Wnt signaling in colorectal cancer. Our results showed that CHD1L expression positively correlates with activated Wnt signaling, and that its expression alone is able to modulate Wnt target gene expression in colorectal cancer cell lines. Overexpression of CHD1L was also found to promote the malignant phenotype in colorectal cancer by upregulating mesenchymal gene expression and promoting cancer stem cell proliferation. Additionally, we found that CHD1L interacts directly with members of the TCF-transcriptional complex. These results indicate that CHD1L is a potential novel druggable target to modulate Wnt signaling. While gene expression and genetic manipulation in vitro experiments showed that CHD1L was involved in Wnt signaling, it still was unknown if CHD1L could be successfully targeted by small molecules. To establish CHD1L as a druggable target, we purified recombinant CHD1L in E. coli, developed a high-throughput screening assay, and screened over 20,000 compounds. A number of hits were identified, and this dissertation details the efforts to validate these small molecules as CHD1L inhibitors. We identified three molecules that inhibited Wnt target gene expression and reversed the malignant phenotype in colorectal cancer cell lines. These results mirrored the observed phenotype after genetic knockdown of CHD1L. Furthermore, these inhibitors showed potent activity in patient-derived tumor organoids (PDTOs) models. PDTOs have been shown to produce predictive clinically relevant responses to drugs, and the identified CHD1L inhibitors outperformed a number of FDA-approved and experimental compounds. From our in vitro experiments we identified one molecule as our lead molecule, referred to in this dissertation as compound 6. We characterized the in vitro and in vivo pharmacokinetics and performed a short-term in vivo pharmacodynamics study. Compound 6 showed good drug-like behavior, reversed EMT gene expression, and induced apoptosis in a preliminary in vivo study. The activity of lead CHD1L inhibitors validates CHD1L as a druggable target in colorectal cancer, and the work in this thesis sets the foundation to develop the next generation of CHD1L inhibitors.