High Cysteinyl Leukotriene Receptor 1 Expression Correlates with Poor Survival of Uveal Melanoma Patients and Cognate Antagonist Drugs Modulate the Growth, Cancer Secretome, and Metabolism of Uveal Melanoma Cells

Abstract

Uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic uveal melanoma, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012) and overall survival (p = 0.011). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05), but significantly upregulated IL-10, IL-1β, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.

Discovery and Development of the Quininib Series of Ocular Drugs

“…The parallel upregulation of VEGF-C and bFGF following treatment with quininib in primary UM cells may be compensatory result of a form of "pseudohypoxia" as previously described in UM and other tumour types (67). Our in vitro data was supported in zebrafish models whereby quininib drugs significantly inhibit the growth of both primary and metastatic zebrafish xenograft models (71).…”

“…In primary and metastatic human UM cell lines (harbouring GNAQ mutations, not the CYSLTR2 oncogene), we observed CysLT1 antagonists (quininib and 1,4-dihydroxy quininib), but not a CysLT2 specific antagonist (HAMI 3379), to significantly alter the survival, long-term proliferation, metabolism, and secretion of inflammatory and angiogenic factors in vitro (71). Interestingly, 24-hour treatment with 20 μM quininib series drugs increases the secreted levels of angiogenic markers in primary and metastatic UM cells (71). In primary UM cells, 20 μM quininib significantly increased secretion of VEGF-C and bFGF, while 20 μM 1,4-dihydroxy quininib significantly increased secretion of Flt-1.…”

“…The quininib series also highlighted a previously unforeseen therapeutic opportunity in UM. Antagonists to CysLT1, but not CysLT2, alter cell survival and long-term proliferation, in UM models (71) supporting antagonism of CysLT1 as a therapeutic strategy in UM.…”

“…Our knowledge of the antiangiogenic properties of the quininib series, coupled with the finding that CYSLTR2 is a UM oncogene, led us to hypothesize that CysLT receptors are a relevant therapeutic target in UM (69). Using the published TCGA dataset (70) and de novo immunohistochemical approaches in primary UM patient samples, we discovered high expression of CysLT1 associated with reduced overall survival and reduced survival from metastatic disease (71). In primary and metastatic human UM cell lines (harbouring GNAQ mutations, not the CYSLTR2 oncogene), we observed CysLT1 antagonists (quininib and 1,4-dihydroxy quininib), but not a CysLT2 specific antagonist (HAMI 3379), to significantly alter the survival, long-term proliferation, metabolism, and secretion of inflammatory and angiogenic factors in vitro (71).…”

“…Using the published TCGA dataset (70) and de novo immunohistochemical approaches in primary UM patient samples, we discovered high expression of CysLT1 associated with reduced overall survival and reduced survival from metastatic disease (71). In primary and metastatic human UM cell lines (harbouring GNAQ mutations, not the CYSLTR2 oncogene), we observed CysLT1 antagonists (quininib and 1,4-dihydroxy quininib), but not a CysLT2 specific antagonist (HAMI 3379), to significantly alter the survival, long-term proliferation, metabolism, and secretion of inflammatory and angiogenic factors in vitro (71). Interestingly, 24-hour treatment with 20 μM quininib series drugs increases the secreted levels of angiogenic markers in primary and metastatic UM cells (71).…”