A phase 0 trial of riluzole in patients with resectable stage III and IV melanoma

Abstract

Purpose: Ectopic expression of GRM1 in murine melanocytes results in transformation into a form of melanoma, and more than 60% of human melanoma samples tested ectopically express GRM1. Stimulation of this receptor in vitro results in up-regulation of activated extracellular signal-regulated kinase (ERK). Furthermore, a xenograft model of melanoma treated with riluzole, an oral GRM1 blocking agent, showed decreased tumor growth compared with the untreated controls. We have now completed a phase 0 trial of riluzole in patients with melanoma.

Experimental design: Patients enrolled on this trial underwent a pretreatment biopsy, took 200 mg of oral riluzole per day for 14 days, and then underwent resection of their remaining tumor. We compared the levels of pERK and pAKT in the pretreatment and post-treatment samples and assessed the metabolic activity of pretreatment and post-treatment tumors using fluorodeoxyglucose positron emission tomography (FDG-PET) scanning.

Results: We accrued 12 patients and all expressed GRM1. We found a significant decrease in pAKT and/or pERK in post-treatment tumor samples as compared with pretreatment samples in 4 (34%) patients. These four patients had a significant decrease in FDG-PET intensity post-treatment as well. Two other patients had a clinical response with no corresponding metabolic response; five patients had similar pretreatment and post-treatment FDG-PET scan findings; and one patient had progressive disease.

Conclusions: Our data show that glutamate blockade with riluzole can inhibit signaling through the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT pathways and suppress the metabolic activity of melanoma. The ectopic expression of metabotropic glutamate receptors may be important in the pathogenesis of human melanoma, and targeting this pathway may be an effective therapy.

Fig. 1

Pretreatment and post-treatment tumor specimens stained for GRM1, Ki-67, pERK, and activated caspase-3 from one of the patients who had a metabolic and radiologic response to riluzole administration (patient 5). There seems to be no change in the pre- and post-staining intensity for GRM1 and Ki-67. There is a decrease in staining intensity for pERK in the post-treatment specimen as compared with the pretreatment specimen. Finally, ~25% of the cells in the post-treatment sections are positive for activated caspase-3 whereas few of the pretreatment tumor cells stain for activated caspase-3.

Fig. 2

A representative FDG-PET scan of one of the four patients that had a radiologic and metabolic response to 2 wk of riluzole administration. Pretreatment FDG-PET scan (bottom) and post-treatment scan (top) done 2 wk after the start of riluzole. There is a large decrease in the number of FDG-avid lymph nodes seen in the post-treatment scan, and the remaining node seen in the top image has decreased in intensity by >40%. The three other patients who had metabolic responses had similar FDG-PET scan results. The far left images show where the pretreatment biopsy was taken.

Fig. 3

Total protein lysates were prepared as indicated in Materials and Methods. A, total protein lysate (10 µg) was resolved by SDS-PAGE, electroblotted onto polyvinylidene difluoride membranes, and probed with the indicated phospho-antibodies. The membranes were then probed with goat anti-rabbit IgG conjugated to horseradish peroxidase. The blots were developed using ECL Advance and exposed to X-ray film. The membranes were then subsequently stripped using Western Reprobe according to the manufacturer's protocol. The membranes were then probed with the indicated protein antibodies and developed as above. The blots are representative of three independent experiments. The band intensities from three independent experiments were quantified using ImageJ 1.41L. Phospho-ERK1/2 (B) and phospho-AKT (C) levels were normalized to their respective protein expression levels. Phosphorylation levels of the posttreatment tumor samples were then compared with the matched pretreatment samples to generate the relative fold increase in phosphorylation.