Abstract
Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.
Highlights
Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases
An irreversible third-generation EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) that is now is used as first-line treatment for patients with EGFR mutation-positive NSCLC16,17, are effective on PC9 at low nanomolar concentrations (Fig. 1a)
After continuous treatment with the erlotinib some subpopulations of cells survive and begin expansion (Fig. 1b and Supplementary Fig. 1a). Such resistance is clinically relevant to non-small-cell lung carcinoma (NSCLC) patients that were treated with EGFR inhibitors[6]
Summary
Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. We use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Immunohistochemical staining and fluorescent in situ hybridization (FISH) in human tissues revealed a wide variation in expression of multiple cancer biomarkers between different cells These routine observations were supported by more advanced methods, such as immunological assays with antibodies revealing pathway activation, functional assays with small-molecule inhibitors, and whole-exome sequencing of matched patient’s samples. Integrative data analysis uncovers mechanisms through which drug tolerance arises in NSCLC cell line models during treatment This pertains to the establishment of distinct drug tolerant states that can co-occur within NSCLC cell populations and express distinct combinations of markers that can be used as prognostic and/or therapeutic targets for small-molecule therapies
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