Abstract 3170: AKT/mTOR pharmacologic inhibition sensitizes RAS/MAPK-activated neuroblastoma cells to MEK1/2 inhibitors
Authors: Yohe Marielle , Lam Norris , Kowalczyk Joshua , Thiele Carol J. 
Published: August 2018

Abstract
Relapsed neuroblastoma tumors have an increased incidence of activating mutations in the RAS/MAPK pathway. Pre-clinical studies indicate these tumors have an increased sensitivity to inhibitors of the RAS/MAPK pathway but single-agent therapeutic approaches have rarely led to durable responses. For this reason, we evaluated MEK1/2 inhibitors, Selumetinib(SEL) and Trametinib(TRAM), for their efficacy in controlling the growth of NB cells alone or in combination with inhibitors of the AKT/mTORi pathways. MYCNamplified [(MYCNamp) KCNRAKLF1174L, KellyALKF1174L,NGPNF1del] or MYCNwild-type[(WT) ASNF1del,NRASQ61K, SY5YF1174L] NB cell lines were used in the study. For tested NB cells, the TRAM IC50 was 10-80fold lower (4-43nM) compared to SEL IC50 (325-525nM). TRAM induced growth inhibition (10nM in AS at 24hrs) was accompanied by an increase in cells in G1. In other tumor types, MEK inhibition is associated with feedback activation of the PI3K/AKT pathway. While we did not see MEKi induced activation of the AKT/mTOR pathway at 1-3 days by Western analysis, combination dose responses (TRAM or SEL +/- MK2206 or Rapamycin) were initiated for each cell line and continued until control-treated cells reached stationary phase using a 384-well format. In SY5Y cells, treatment with MK2206 (allosteric Akt1/2/3 inhibitor) weakly inhibited cell proliferation (IC50 ~ 250nM). Using the Bliss Independence model, we computationally elucidated doses of MEK1/2i and Akt/mTORi that resulted in the greatest synergy in suppressing cell proliferation. MK2206(200nM) increased the sensitivity of SY5Y cells to TRAM some 2.5fold as the TRAM IC50 decreased from 35 to 13nM with MK2206. Using the Bliss independence model, combination of 200nM MK2206 and 0.01µM TRAM showed the greatest synergy in inhibiting cell numbers. MK2206(200nM) increased the sensitivity of SY5Y cells to SEL ~37-fold as the SEL IC50 decreased from 4.3 to 13µM with MK2206. MK2206(200nM) with 0.37µM SEL showed the greatest synergy in inhibiting cell growth. Similar results were also seen in MYCNamp Kelly NB cells. Next, we determined whether similar sensitization to MEK1/2i could be seen with treatment with the mTORi rapamycin. As a single agent SY5Y and Kelly NB cells are insensitive to rapamycin (1.6nM) but in combination with1.6nM rapamycin in combination with 40nM TRAM synergistically inhibited cell growth. In Kelly cells, 1.6nM rapamycin (insensitive as a single agent) and 330nM TRAM synergistically inhibited cell growth. In conclusion, using the Bliss independence model for synergy, we found that Akt or mTOR inhibition synergized with MEK inhibitors in neuroblastoma cell lines with activating RAS or ALK mutations.
Citation Format: Marielle Yohe, Norris Lam, Joshua Kowalczyk, Carol J. Thiele. AKT/mTOR pharmacologic inhibition sensitizes RAS/MAPK-activated neuroblastoma cells to MEK1/2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3170.