Abstract P066: Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer

Abstract

Drug resistance is ultimately the cause of death for most cancer patients – even initially strong responses to treatment are usually followed by the emergence of resistance over time. This suggests the existence of residual or persistent cancer cells, creating a reservoir that ultimately gives rise to stable resistance. These drug tolerant persisters (‘the deadly survivors’) have been described for over a decade in numerous studies; they are often present as a minor fraction of the total tumour population and may exploit non-genetic (transcriptional) programs to allow the cells to survive drug treatment. 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. We carried out parallel genome-wide CRISPR gain and loss of function screens in EGFR mutant lung cancer cell lines treated with EGFR inhibitors, to identify the genes and pathways that may be important in enabling the survival of persister cells. We observed recurrent resistant genes in previously identified resistance pathways including PI3K (PTEN, TSC1, TSC2), MAPK (KRAS, NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, KEAP1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockout demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, key activators of the Hippo pathway. A closer review of the CRISPR screen data confirmed that many resistance hits are members of this pathway - upstream regulators (NF2, AMOTL2), core signalling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signalling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and increased expression of canonical Hippo transcriptional targets. We therefore reasoned that the Hippo pathway might be involved in maintaining the survival of drug tolerant persister cells in this setting. Acute treatment of EGFR mutant cell lines with osimertinib was associated with increased nuclear localisation of YAP1 and WWTR1 and increased expression of canonical Hippo transcriptional targets. Furthermore, the combination of osimertinib and a TEAD inhibitor (K-975) almost completely abolished the survival of drug tolerant persister cells following treatment, indicating that this pathway is an important survival mechanism following drug treatment. Consequently, we propose Hippo signalling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Ercia Banks, Nin Guan, Shanade Dunn, Maria Lisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P066.

Targeting YAP1/TAZ in nonsmall‐cell lung carcinoma: From molecular mechanisms to precision medicine

“…Treating these cells with YAP1 inhibitor XAV‐939 or TEAD inhibitor MYF‐01‐037, together with MEK inhibitor trametinib, resensitizes these cells to the EGFR‐TKI osimertinib. Treating these cells with MYF‐01‐037, trametinib and osimertinib also precipitously reduced the number of dormant cells, compared to not using the TEAD inhibitor 139,140 …”

“…YAP1 is found to repress transcription of the proapoptotic protein BMF via the YAP/TEAD/Slug complex, aiding these cells to evade apoptosis following EGFR and MEK inhibition.Treating these cells with YAP1 inhibitor XAV-939 or TEAD inhibitor MYF-01-037, together with MEK inhibitor trametinib, resensitizes these cells to the EGFR-TKI osimertinib. Treating these cells with MYF-01-037, trametinib and osimertinib also precipitously reduced the number of dormant cells, compared to not using the TEAD inhibitor 139,140. 6 | CONCLUSION AND FUTURE DIRECTIONFollowing accumulating evidence of Hippo-YAP1/TAZ pathway in tumorigenesis, the roles of YAP1/TAZ in NSCLC are becoming apparent.…”