Abstract 377: Novel mTOR inhibitor MLN0128 inhibits imatinib-resistant GIST more potently than rapalogues by abrogating AKT and 4EBP1 activation
Authors: Mühlenberg Thomas , Ketzer Julia , Fletcher Jonathan A. , Bauer Sebastian 
Published: July 2016

Abstract
Introduction:
Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of the KIT or PDGFRA receptor tyrosine kinases. Most patients respond to the KIT/PDGFRA inhibitor imatinib (IM) but eventually progress due to secondary resistance mutations in KIT. Development of salvage treatments is hampered by the genomic heterogeneity of resistance mutations as single KIT-inhibitory drugs do not inhibit all mutants. The PI3K pathway is strongly activated in GIST regardless of secondary KIT mutations and thus provides a rational target for therapeutic combinations. Rapamycin-analogues have yet shown limited clinical benefit in GIST but next-generation mTOR inhibitors have not been tested in GIST. Here we evaluated the effects of the novel mTOR inhibitor MLN0128 alone and in combination with other kinase inhibitors in GIST.
Methods:
Three IM-sensitive (IM-S; GIST-T1, GIST882, GIST430) and 2 IM-resistant (IM-R; GIST430/654, GIST48B) cell lines were studied. Cells were treated with MLN0128 alone and in combinations with KIT inhibitors (IM, sunitinib, regorafenib), MEK1/2 inhibitor trametinib, and everolimus. Cell viability was evaluated by Sulforhodamin B (SRB) assay after 3 or 6 days of treatment. Biological consequences of on KIT, KIT-dependent signaling intermediates and apoptosis were evaluated by immunoblotting.
Results:
In cell viability assays MLN0128 displayed IC50 values between 15nM (GIST-T1) and 30nM (GIST48B). Immunoblots revealed inhibition of phosphorylation of ribosomal protein S6 (as marker for mTOR activation) at 1nM with complete inhibition at 50-100nM in all cell lines. In contrast to everolimus, MLN0128 did also abrogate phosphorylation of 4E-BP1 at 50-100nM. At these concentrations we also observed strong inhibition of AKT phosphorylation (except in GIST48B), upstream of mTOR, while phosphorylation of KIT remained unchanged. A notable induction of ERK1/2 phosphorylation in response to MLN0128 treatment suggested a feedback loop activating the MEK/ERK signaling. Combinations of MLN0128 with the clinical MEK1/2 inhibitor trametinib as well as with different KIT inhibitors exhibited additive effects.
Conclusions:
MLN0128 has strong antiproliferative effects in IM-sensitive and IM-resistant cell lines in the low nanomolar range, including KIT-negative GIST. The distinct inhibitory profile, inhibition of AKT as well as 4E-BP1, suggests superior clinical efficacy compared to first- and second-generation rapalogues. Combinations with approved KIT and MEK inhibitors display additive effects and may represent a feasible clinical strategy, which warrants further investigation.
Citation Format: Thomas Mühlenberg, Julia Ketzer, Jonathan A. Fletcher, Sebastian Bauer. Novel mTOR inhibitor MLN0128 inhibits imatinib-resistant GIST more potently than rapalogues by abrogating AKT and 4EBP1 activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 377.