RAS regulators are made up of two classes: (1) GTPase-activating proteins (GAPs), which inactivate RAS by stimulating GTP hydrolysis, and (2) guanine nucleotide exchange factors (GEFs), which activate RAS by catalysing the release of GDP. Previous work has shown that RASAL2 (a RAS GAP) confers resistance to chemotherapy in patients with triple negative breast cancer (TNBC). Here, I found that RASAL2 also confers resistance to PARP inhibitors (PARPi) in TNBC cells. Furthermore, quantification of TNBC patient specimens shows that RASAL2 is enriched in tumour tissues versus adjacent non-malignant tissues, suggesting that RASAL2 is a tumour-specific factor. To test whether RASAL2 could be a drug target, I conducted short-term (3-day) drug screening assays on an isogenic cell-based model of RASAL2-expressing TNBC cells (MDA-MB-468), using 19 drug-like chemical compounds that have been predicted to have the structural ability to interact with RASAL2. My screening identified 14 compounds with significantly differential sensitivities in RASAL2-expressing cells, implying that these compounds modulate RASAL2 activity. Next, I selected a subset of the compounds and validated my short-term drug screening results with long-term (10-day) clonogenic assays. I also evaluated their ability to affect RAS GTPase activity using enzyme-linked immunosorbent assay. Preliminarily, my data showed little change in RAS GTPase activity following exposure to the compounds at 1 μM, suggesting that at this concentration RAS signalling was not perturbed. Finally, using human patient and cell line data, my exploratory in-silico analyses identified RASGRP1 (a RAS GEF) as a RAS regulator associated with therapy resistance. Together, my project shows that RAS regulators are likely to be implicated in modulating therapy response in TNBC. Future work could focus on the underlying mechanisms associated with the expression of these regulators.
Investigating the therapeutic implications of RAS Regulators in Triple Negative Breast Cancer
O'Connor, C. E. (Author). 19 Mar 2024
Student thesis: Master's Thesis › Master of Science by Research (MScR)