Abstract 5823: Modulation of the Hippo pathway protein YAP as a mechanism of resistance to MEK inhibition in MAPK hyperactivated neuroblastoma

Background: Current therapies for high-risk neuroblastoma are highly toxic and ineffective, as a majority of patients will face therapy-resistant relapses. Our recent whole genome sequencing analysis revealed that a majority of relapsed tumors had mutations causing RAS-MAPK pathway activation. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP in relapsed neuroblastoma as well as resistance to MEK inhibition in other cancer models. Based on these findings, we hypothesized that YAP activity may lead to acquired resistance to MEK inhibition, and that dual inhibition of these two pathways may have synergistic therapeutic efficacy. Methods: We selected the cell line NLF from a panel of NB cell lines due to its high YAP expression and NF1 loss. NLF cells were treated with MEK inhibitor trametinib (20 nM) over a 72-hour period and whole cell lysates and nuclear lysates were collected for immunoblot. Effects of YAP knockdown via siRNA transfection with and without trametinib co-treatment on survival were assessed by CellTiter-Glo and RT-CES. Cell signaling analyses of tumor lysates from mouse SKNAS (NRAS Q61K) xenografts treated with vehicle, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg for YAP, ERK, and apoptosis pathways were performed via immunoblot. Results: Immunoblots of 72-hour time course treatments of trametinib in NLF showed decreased pYAP, the cytosolic inactive form of the YAP protein, indicating increased YAP capable of translocating to the nucleus, which was confirmed by analyses of nuclear extracts. siRNA-mediated knockdown of YAP alone in NLF cells had a modest effect on survival, while combinatorial inhibition of MEK and YAP signaling caused a significant reduction in cellular proliferation and survival. Immunoblotting of trametinib-treated SKNAS xenografts indicated decreased pYAP and pERK in response to increasing trametinib dose, while total YAP levels remained constant. Conclusion: Our results demonstrate that prolonged trametinib exposure causes a marked nuclear enrichment of unphosphorylated YAP and co-inhibition of MEK and YAP in neuroblastoma models effectively reduces proliferation and survival. We are currently investigating the effects of MEK inhibition on the expression of YAP target genes and on cellular proliferation upon modulation of YAP protein expression. Note: This abstract was not presented at the meeting. Citation Format: Grace E. Coggins, Lori S. Hart, Jimmy Elias, John M. Maris. Modulation of the Hip