Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are highly effective therapies for sub-populations of non-small cell lung cancers. Specific mutations have been identified in the EGFR gene such as L858R which overstimulate cell pathways that lead to tumor growth. All tumors eventually develop resistance to this treatment, rendering them useless, and tumor growth progresses. Escape mutations in the EGFR gene were first seen in patients undergoing treatment with first-generation TKI erlotinib and gefitinib. T790M is a widely seen gate-keeping mutation which overcomes inhibition from erlotinib and gefitinib. Third-generation irreversible TKI, osimertinib, can inhibit tumor cells with this gate-keeping mutation thus overcoming a major hurdle in containment of tumor growth. Unfortunately, patients eventually develop resistance to osimertinib, exhausting options for managing non-small cell lung cancer. Here we analyzed H1975 cells which harbor L858R + T790M mutations. We aimed to track genomic, transcriptomic, and proteomic changes to uncover mechanisms cells use to develop resistance to osimertinib.
We established cell colonies which were able to survive high dose treatment up to 2 µM osimertinib. We also saved cells with IC50 of 30 nM to represent drug-tolerant cells. We conducted single-cell sequencing of mRNA transcription and performed hierarchal gene analysis which identified CD74 as a novel factor which was upregulated in drug-tolerant cells. Further we showed CD74 gene was accessible as open chromatin for easy upregulation. Western blot analysis showed increased expression of CD74 after 24 hours of osimertinib treatment. Using siRNA in H1975 cells, we conducted knockdown experiments of CD74 during osimertinib treatment and showed reduced viability. Next, H1975 cells lines were engineered with deletions in CD74 to knockout its expression. These cells also showed reduced viability in the presence of osimertinib. Quantification of apoptosis using caspase-glo assays showed greater activation of apoptosis in cell populations without CD74 compared to normal H1975 cells. H1975-CD74 knockout cells also took longer to become resistant to osimertinib when compared with control. These results show the role of CD74 in helping tumor cells survive EGFR TKI treatment. / 2023-12-05T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43520 |
| Date | 06 December 2021 |
| Creators | Plotnick, David O. |
| Contributors | McKnight, C. James |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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