Abstract A117: The epigenome and gene expression landscape of osimeritib drug tolerant persister cells
Authors: Criscione Steven W , Martin Matthew , Yao Yi , Markovets Aleksandra , Zhang Jingwen , Gagrica Sladjana , Delpuech Oona , Lai Zhongwu , Smith Paul , Dry Jonathan 
Published: March 2020

Abstract
Osimertinib is a potent EGFR inhibitor (EGFRi) that is approved to treat patients with metastatic non-small cell lung cancer (NSCLC) with EGFR gene mutations. Despite the benefit observed for osimertinib many patients still develop resistance and relapse on treatment. One mechanism thought to cause resistance is de novo acquisition of resistant molecular changes in a drug tolerant persister (DTP) cell population. Here we characterized the molecular phenotypes of osimertinib DTPs to identify novel vulnerabilities to eliminate DTPs and prevent the emergence of drug resistance. We used a multiomics approach by combining transcriptomics (RNA-seq), proteomics (reverse phase protein array), and epigenomics assays (ATAC-seq) to profile osimertinib DTPs in four EGFRm NSCLC cell-lines (PC9, H1975, HCC2935, and HCC827). We compared DTPs generated by three weeks exposure to osimertinib to DMSO control, 24 hrs. acute treatment, and DTPs removed from drug. Due to the nature of the time-course we could disentangle effects of acute treatment from long-term DTPs; for example, we observed downregulation of cell-cycle and cell-cycle regulated processes in both acute treatment and DTPs. Phenotypically DTPs were in a non-proliferative state, morphologically larger, and resistant to apoptosis. Unsupervised clustering of multiomics data revealed that DTPs are distinct from acute treated and DMSO control cells at the epigenome, RNA, and protein-level. Interestingly, DTPs removed from drug for up to seven days do not fully return to control state but instead maintain some features in common with DTPs despite regaining proliferative ability. Motif analysis of regions that increased chromatin accessibility in Osimertinib DTPs identified an enrichment for TEAD transcription factor binding sites. TEAD transcription factors are regulated by the transcription co-activators YAP1/TAZ. When the Hippo pathway is on YAP1/TAZ is phosphorylated and localized to the cytoplasm, whereas when the pathway turns off YAP1/TAZ loses phosphorylation, translocate to the nucleus, and acts as a co-factor for TEAD transcription factors. We validated by Western blot that in osimertinib DTPs YAP1 loses phosphorylation indicating that the Hippo pathway switches from on to off. YAP1/TEAD downstream target genes also increase RNA expression in osimertinib DTPs. In addition to the Hippo pathway switch, and potentially related, we observe epithelial to mesenchymal (EMT) transition as a prominent molecular phenotype of DTPs across all four models. The EMT transition was evident at the transcriptome, proteome, and epigenetic level and is a prominent feature of surviving cells but not the 24 hrs. acute treatment. The chromatin accessibility changes at EMT regulated genes suggest the possibility that the EMT transition may be regulated by an epigenetic switch. Consistent with this idea we demonstrate that multiple drugs targeting the epigenome including HDAC inhibitors can effectively block regrowth of DTPs after osimertinib removal. Together our data suggests blocking protective changes to the chromatin landscape may represent a therapeutic strategy to target osimertinib DTPs.
Citation Format: Steven W Criscione, Matthew Martin, Yi Yao, Aleksandra Markovets, Jingwen Zhang, Sladjana Gagrica, Oona Delpuech, Zhongwu Lai, Paul Smith, Jonathan Dry. The epigenome and gene expression landscape of osimeritib drug tolerant persister cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A117. doi:10.1158/1535-7163.TARG-19-A117