(5-(2-4-bis((3s)-3-methylmorpholin-4-yl)pyrido(2-3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol and Uveal-Neoplasms

Over 90% of uveal melanomas harbor pathogenic variants of the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were vulnerable to a combination of survival pathway inhibitors and the histone-deacetylase inhibitor romidepsin, we investigated whether this combination would be effective in models of uveal melanoma.. A small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4; OTX-015), extracellular signal-related kinase (ERK; ulixertinib), mechanistic target of rapamycin (mTOR; AZD-8055), or phosphoinositide 3-kinase (PI3K; GDC-0941) combined with a clinically relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92.1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hours. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, AZD-8055 alone, or the combination, and protein changes were validated by immunoblot.. AZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequencing analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055-treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 transcripts noted in the sequencing analysis were confirmed at the protein level in Mel202 cells.. Our data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.

Topics: Apoptosis; Bcl-2-Like Protein 11; bcl-X Protein; Caspase 3; Cell Line, Tumor; Depsipeptides; Drug Combinations; Flow Cytometry; Gene Expression Regulation, Neoplastic; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Immunoblotting; Melanoma; Morpholines; Poly(ADP-ribose) Polymerases; Retinal Pigment Epithelium; Sequence Analysis, RNA; Survivin; TOR Serine-Threonine Kinases; Uveal Neoplasms

Uveal melanoma (UM) is an intraocular tumor which is almost lethal at the metastatic stage due to the lack of effective treatments. In this regard, we have developed an albumin-based nanostructure (ABN) containing AZD8055 (ABN-AZD), which is a potent mTOR kinase inhibitor, for its efficient delivery to the tumors. The drug has been conjugated to ABN using tailored linkers that have a disulfide moiety, allowing its release selectively and effectively in the presence of an elevated concentration of glutathione, such as inside the tumoral cells. Our therapeutic approach induced significant cellular toxicity in uveal melanoma cells, but not in non-tumoral keratinocytes, highlighting the excellent selectivity of the system. In addition, these nanostructures showed excellent activity in vivo, decreasing the tumor surface compared to the free AZD8055 in mice models. Remarkably, the results obtained were achieved employing a dose 23 times lower than those used in previous reports.

Topics: Animals; Feeder Cells; Humans; Melanoma; Mice; Mice, Nude; Morpholines; Nanostructures; Neoplasm Proteins; Serum Albumin, Human; TOR Serine-Threonine Kinases; Uveal Neoplasms; Xenograft Model Antitumor Assays

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Benzimidazoles; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Genotype; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Mechanistic Target of Rapamycin Complex 2; Melanoma; Mice; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Morpholines; Multiprotein Complexes; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 1; TOR Serine-Threonine Kinases; Uveal Neoplasms; Xenograft Model Antitumor Assays