Despite recent advancements in cancer treatment, the management of heterogeneous
cancers poses a persistent challenge. This is best exemplified by the limited success in treating
acute myeloid leukemia (AML), which still exhibits a 5-year survival rate of less than 30% to date. The resistance to treatment and high likelihood of relapse in AML patients is believed to be
attributed to the elusive nature of cancer stem cells (CSCs). CSCs are characterized by their self-renewal capabilities and blockade to normal differentiation into mature hematopoietic cells,
however they are only functionally defined through their engraftment potential upon
transplantation into immunodeficient mice to phenocopy human tumorigenesis. Therefore
isolation, study and treatment of CSCs is a prominent challenge in cancer research. To circumvent this challenge, our research group has developed a proprietary screening platform capable of identifying compounds within vast chemical space that selectively target surrogate human CSCs while preserving healthy stem cell counterparts. Our approach of screening and identifying compounds that selectively target human CSCs compared to normal SCs focuses on custom compound libraries curated through pre-screening for anti-proliferative and lethality properties in Escherichia coli (E. coli). From a pool of 21,000 active molecules selected from several libraries chemical space totalling 8.7 million compounds, we identified 15 distinct structural frameworks or "scaffolds", each representing different chemical structures that form the basis for potential drug development. Through secondary assays using primary AML patient derived cells, we have prioritized a novel compound, termed MLMB-2231 as a promising lead anti-CSC candidate. However, beyond MLMB-2231's demonstrated ability to selectively target human CSCs over normal stem cells, the cellular and molecular mechanisms of activity are unknown, limiting improvements and use towards investigational new drug application (IND) initiation. Here, I have utilized a variety of chemical genomics techniques to probe the downstream effects and gene targets of MLMB-2231. Apoptosis and cell cycling assays demonstrated that MLMB-2231 operates through an induction of apoptosis at 48h resulting in a G0/G1 cell cycle stall. The use of genome-wide CRISPR viability screening combined with transcriptomic analysis through RNA sequencing identified significant upregulation of pathways associated with aberrant ubiquitin- protease system (UPS), disruption of cell cycling, and upregulation of apoptotic pathways. These findings suggest that MLMB-2231 exerts its effects by interfering with the UPS, leading to impaired protein degradation and cellular stress, disrupting key cell cycle checkpoints, and inducing apoptosis. Future studies will focus on hit validation to confirm direct binding targets and incorporate animal models to evaluate pharmacokinetics (pK) and overall efficacy in vivo, paving the way for potential clinical applications. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/29986 |
| Date | January 2024 |
| Creators | Shull, Caylie |
| Contributors | Bhatia, Mick, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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