Abstract 5082: Pre-clinical study targeting metabotropic glutamate receptor (GRM1) angiogenesis pathway.
Authors: Grabell Daniel A. , Wen Yu , Semlani Neha , Li Jiadong , Goydos James 
Published: August 2014

Previously our group reported the identification of metabotropic glutamate receptor (GRM1) as an oncogene and as a potential therapeutic target of melanoma. We reported two clinical trials of Riluzole, an FDA approved glutamate release inhibitor for ALS, in advanced melanoma patients that showed suppression of PI3K/AKT and MAPK pathway and demonstrated clinical response of 34%. Recently, our lab found that over expression of GRM1 in a low GRM1-expressing melanoma cell line UACC903 GRM1+, BRAFV600E, PTENMUT, stimulates angiogenesis through activating the signaling pathway of AKT-mTOR-HIF1-IL8, VEGF even under the normoxia condition. We also demonstrated that Riluzole inhibits angiogenesis through the signaling pathway of GRM1/AKT/HIF1 among the patients responding to Riluzole in our phase II clinical trial. To facilitate translating the discovery of GRM1 stimulating angiogenesis into clinic, we designed an in vitro pre-clinical study assessing the combinatory effects of Riluzole with the agents targeting the angiogenesis pathway of GRM1.

We tested an AKT inhibitor, MK-2206 and a HIF1 inhibitor, Aminoflavone, the active component of AFP464, which is in Phase I clinical trial and a ligand probably activating the aryl hydrocarbon receptor (AhR) in hypoxia preventing angiogenesis, even though it is reported that Aminoflavone also works in an AhR-independent fashion reducing HIF-1alpha in certain cell lines. Five melanoma cell lines UACC903GRM1 +,BRAFV600E,PTENMUT, SKMel2GRM1+,NRASQ61R,PTENWT,C8161GRM1+,BRAF/NRAS/PTENWT,A2058GRM1+,BRAFV600E,HT144GRM1+,BRAFV600E,PTENMUT were tested for HIF1 expression. HT-144, a BRAF mutant that is a V600E homozygous line which is particularly aggressive, showed highest expression of HIF1 alpha through western blotting. The AKT inhibitor and the HIF1 alpha inhibitor were tested alone and in combination with Riluzole in HT-144 cell line. Cell proliferation response to the treatments was assessed through MTS assays. HIF-1 and pAKT inhibition was assessed through Western Blots and relative mRNA level of HIF1 and IL-8 was quantified using qRT-PCR.

The monolayer growth assays demonstrated significant inhibition of growth with combination treatments versus single treatments at hypothesized clinically beneficial dosage. Also, MK-2206 and Aminoflavone when combined with Riluzole led to larger decrease in HIF and pAKT levels relative to DMSO treated cells at protein level. HT144 cells also demonstrated angiogenesis signaling response to the combination treatment of Riluzole with MK-2206 or Aminoflavone, which was observed by the significant inhibition of IL8 and Hif 1 at mRNA level. We conclude that HIF1 interacts with GRM1 in stimulating angiogenesis and the combination of glutamate release inhibitor with the agents targeting the downstream signaling of GRM1 could provide a better clinical outcome for melanoma patients as compared to single agent Riluzole.

Citation Format: Daniel A. Grabell, Yu Wen, Neha Semlani, Jiadong Li, James Goydos. Pre-clinical study targeting metabotropic glutamate receptor (GRM1) angiogenesis pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2013-5082