Abstract PO-103: Characterizing epigenetic and genetic mechanisms of glioma drug resistance using simultaneous lineage tracing and single-cell transcriptomic analysis
Authors: Eyler Christine E. , Matsunaga Hironori , Hovestadt Volker , Vantine Samantha J. , van Galen Peter , Bernstein Bradley E. 
Published: December 2020

Therapeutic pressure can result in tumor evolution and adaptation via genetic and epigenetic mechanisms, which may be transient or stable. Knowledge of how these events contribute to intratumoral heterogeneity and dynamic subpopulations will require single cell-resolution experimental approaches that prospectively label, longitudinally track, and functionally characterize adaptive subclonal populations. In glioblastoma, poor efficacy of therapies targeting receptor tyrosine kinases (RTKs) can be attributed to a variety of mechanisms including intrinsic genetic subclonal diversity and therapy-derived epigenetic transitions. To more fully elucidate the factors contributing to RTK inhibitor resistance in glioblastoma, we designed and employed an experimental approach that combines high diversity cell lineage barcoding with single cell transcriptomic analysis. When a PDGFRA-amplified glioblastoma stem cell (GSC) model was treated with the RTK/PDGFRA inhibitor dasatinib, we observed that a broad spectrum of subclones adopt a temporary, Notch-dependent persister phenotype that presumably sustains cells through the initial drug exposure phase. Continued exposure to dasatinib resulted in the outgrowth of rare subclones with preexisting or acquired genetic changes. Single-cell analyses revealed several different subclones bearing distinct copy number amplifications of the insulin receptor substrate-1 and -2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs and have not yet been previously identified as genes involved in glioblastoma drug resistance. Analysis of the Cancer Genome Atlas (TCGA) database revealed that IRS copy number amplifications are evident in a subset of a range of primary tumors and are associated with poor prognosis in glioblastoma subtypes bearing PDGFRA-amplified tumors. Functional characterization confirms that IRS2-amplified subclones lose dependence on the Notch signaling that characterizes the persister phenotype, and instead become dependent on insulin receptor/AKT signaling. Overall, our method for combined lineage-tracing and scRNA-seq revealed interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model. Moreover, this method permitted identification of previously unappreciated genetic alterations that may underlie the inefficacy of targeted therapies in glioblastoma.

Citation Format: Christine E. Eyler, Hironori Matsunaga, Volker Hovestadt, Samantha J. Vantine, Peter van Galen, Bradley E. Bernstein. Characterizing epigenetic and genetic mechanisms of glioma drug resistance using simultaneous lineage tracing and single-cell transcriptomic analysis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-103.