Single-cell lineage analysis reveals genetic and epigenetic interplay in glioblastoma drug resistance

Drug resistance is a central problem in cancer therapy, yet the underlying mechanisms remain obscure. Cancer cells typically acquire resistance via genetic mutations, but may also adopt reversible epigenetic phenotypes that allow them to persist through drug exposure. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade. Overall design: Here we combined cell lineage barcoding and single-cell transcriptomics to trace the emergence of resistance in stem-like glioblastoma (GBM) cells treated with RTK inhibitors. Whereas a broad spectrum of barcoded lineages adopts a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses revealed that these genetic subclones gain copy number amplifications of the IRS1 or IRS2 loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas, and may underlie the inefficacy of targeted therapies in this disease.