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

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

Development of small molecular inhibitors against BRAF and MEK has been a breakthrough in the treatment of malignant melanoma. However, the long-term effect is foiled in virtually all patients by the emergence of resistant tumor cell populations. Therefore, mechanisms resulting in the acquired resistance against BRAF and MEK inhibitors have gained much attention and several strategies have been proposed to overcome tumor resistance, including interval treatment or withdrawal of these compounds after disease progression.. Using a panel of cell lines with an acquired resistance against MEK inhibitors, we have evaluated the sensitivity of these cells against compounds targeting AKT/mTOR signaling, as well as novel ERK1/2 inhibitors. Furthermore, the effects of withdrawal of MEK inhibitor on migration in resistant cell lines were analyzed.. We demonstrate that withdrawal of BRAF or MEK inhibitors in tumor cells with an acquired resistance results in reactivation of ERK1/2 signaling and upregulation of EMT-inducing transcription factors, leading to a highly migratory and invasive phenotype of cancer cells. Furthermore, we show that migration in these cells is independent from AKT/mTOR signaling. However, combined targeting of AKT/mTOR using MK-2206 and AZD8055 efficiently inhibits proliferation in all resistant tumor cell lines analyzed.. We propose that combined targeting of MEK/AKT/mTOR or treatment with a novel ERK1/2 inhibitor downstream of BRAF/MEK suppresses proliferation as well as migration and invasion in resistant tumor cells. We provide a rationale against the discontinuation of BRAF or MEK inhibitors in patients with an acquired resistance, and provide a rationale for combined targeting of AKT/mTOR and MEK/ERK1/2, or direct targeting of ERK1/2 as an effective treatment strategy.

Topics: Benzimidazoles; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; Heterocyclic Compounds, 3-Ring; HT29 Cells; Humans; Melanoma; Melanoma, Cutaneous Malignant; Mitogen-Activated Protein Kinase Kinases; Morpholines; Neoplasm Invasiveness; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6; Signal Transduction; Skin Neoplasms; TOR Serine-Threonine Kinases

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

(V600E)B-RAF mutation is found in 50% to 60% of melanomas, and the novel agents PLX4032/vemurafenib and GSK2118436 that inhibit the (V600E)B-RAF kinase achieve a remarkable clinical response rate. However, as might be expected, acquired clinical resistance to these agents arises in most melanoma patients. PLX4032/vemurafenib resistance that arises in vivo in tumor matched short-term cultures or in vitro in melanoma cell lines is not caused by acquisition of secondary mutations in (V600E)B-RAF but rather is caused by upregulating platelet-derived growth factor receptor β (PDGFRβ) or N-RAS which results in resistance or sensitivity to mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK) kinase inhibitors, respectively. In this study, we define a targeted combinatorial strategy to overcome PLX4032/vemurafenib resistance in melanoma cell lines or short-term culture where the resistance is driven by PDGFRβ upregulation, achieving synergistic growth inhibition and cytotoxicity. PDGFRβ-upregulated, PLX4032-resistant (PPRM) cell lines show dual phospho (p)-ERK and p-AKT upregulation, and their growth inhibitory responses to specific small molecule inhibitors correlated with p-ERK, p-AKT, and p-p70S6K levels. Coordinate inhibition of (V600E)B-RAF inhibition and the RTK-PI3K-AKT-mTORC axis led to functionally significant rebound signaling, illustrating a robust and dynamic network connectivity. Combined B-RAF, phosphoinositide 3-kinase (PI3K), and mTORC1/2 inhibition suppressed both immediate early and delayed compensatory signaling, resulting in a highly synergistic growth inhibitory response but less efficient cytotoxic response. In contrast, the combination of MEK1/2, PI3K, and mTORC1/2 inhibitors consistently triggered apoptosis in a highly efficient manner. Together, our findings offer a rational strategy to guide clinical testing in preidentified subsets of patients who relapse during treatment with (V600E)B-RAF inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Imidazoles; Indoles; MAP Kinase Kinase Kinases; Mechanistic Target of Rapamycin Complex 1; Melanoma; Morpholines; Multiprotein Complexes; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Point Mutation; Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Quinolines; Receptor, Platelet-Derived Growth Factor beta; Recombinant Fusion Proteins; Signal Transduction; Spheroids, Cellular; Sulfonamides; TOR Serine-Threonine Kinases; Transcription Factors; Vemurafenib