Date of Award

Fall 2024

Document Type

Open Access Dissertation

Department

College of Pharmacy

First Advisor

Phillip Buckhaults

Abstract

Precision medicine is a revolutionary approach of targeting genetic alterations that are present only in tumor cells to find vulnerabilities that can be targeted with therapy. Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens can reveal all genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs.

In this dissertation, I comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. I identified all somatic mutations, all expressed genes, and conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. I found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets, genetically and pharmacologically, that could be exploited as treatments for this specific subtype of colon cancer.

Additionally, I investigated three-way synthetic lethal interactions to discover therapeutic targets to re-sensitize resistant TP53 mutant colon cancers to cisplatin. I used a TP53-KO patient-derived colon tumor organoids model, previously subjected to genome-wide CRISPR KO screening, and identified novel targets which are involved in DNA repair pathways, including the Fanconi Anemia (FA) pathway, and their depletion is associated with re-sensitizing TP53 mutant colon cancer organoids to cisplatin.

Finally, I designed a novel multiplex organoid avatar drug screening platform, called StarTrace, which simultaneously test pools of multiple patients’ organoid avatars for sensitivity or resistance to small molecule inhibitors. I utilized both quantitative real-time PCR-based and single-molecule sequencing assays to track the relative Darwinian fitness of each barcoded organoid within the pool. This novel (IP number) assay offers a rapid, cost-effective and sensitive approach for high-throughput cohort testing for personalized treatment. In summary, the data presented in this dissertation identify potential novel therapeutic targets for treating colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.

Rights

© 2025, Sana Khalili

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