Abstract 2886: The Hippo pathway protein YAP mediates resistance to MEK1/2 inhibition in neuroblastoma

Abstract

Abstract Background: Relapsed NBs harbor an enrichment in mutations activating RAS-MAPK signaling pathway. The MEK1/2 inhibitor trametinib has shown tumor growth delay but not regressions in NB preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP in relapsed NBs as well as trametinib resistance in other cancers. We hypothesized that increased YAP transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven NBs. Methods: We screened a panel of NB cell lines for mutations predicted to hyperactive the RAS pathway and for high YAP expression. NLF and SK-N-AS cells were treated with the MEK1/2 inhibitor trametinib (20 nM) over 72hrs, and changes in YAP protein expression and cellular localization were observed via immunoblot. YAP protein expression was modulated in NLF, SK-N-AS, and SK-N-FI cells by siRNA-mediated knockdown, CRISPR-Cas9 genetic knockout, and constitutive overexpression. Effects on survival, proliferation, and signaling were observed with and without trametinib treatment by CellTiter-Glo cell cycle analysis, RT-qPCR, and immunoblot. Synergy studies were performed to assess the combination of trametinib and a YAP/TEAD transcriptional activity inhibitor (Vivace Therapeutics) using Incucyte and CellTiter-Glo. Results: In nuclear extracts of NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K), trametinib caused a near-complete translocation of YAP protein into the nucleus after 72 hrs. Transient knockdown and genetic knockouts of YAP in untreated NLF cells had a modest effect by decreasing proliferation by 20% (p<0.01), while combinatorial inhibition of MEK and YAP signaling caused a significant reduction in cellular proliferation by 70% (p<0.001). Overexpression of the constitutively-active form of the YAP protein, YAP-5SA, rescued this effect. Finally, synergy assays between trametinib and a YAP-TEAD transcriptional activity inhibitor produced combination index values< 1, indicating strong synergy. Conclusion: Prolonged trametinib exposure causes a marked nuclear enrichment of active YAP, and co-inhibition of MEK and YAP in NB models effectively reduces proliferation and survival. Genetic depletion of YAP in NB cell lines caused a significant increase in sensitivity to trametinib, which was rescued with overexpression of a constitutively-active YAP-5SA. We have identified a potential drug combination to target a YAP-driven mechanism of trametinib resistance in RAS-driven NBs to support future development of a novel combination therapy for NB patients. Citation Format: Grace E. Coggins, Alvin Farrel, Komal Rathi, Colin M. Hayes, Tracy Tang, Leonard Post, John M. Maris. The Hippo pathway protein YAP mediates resistance to MEK1/2 inhibition in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2886.