gx-15-070 and Melanoma

MEK kinase inhibitors (trametinib and selumetinib) or kinase inhibitors directed against mutated BRAF(V600E) (vemurafenib and dabrafenib) have initial encouraging effects in the treatment of melanoma but acquired resistance appears almost invariably after some months. Studies revealed mutually exclusive NRAS and BRAF activating mutations driving the MAPK/ERK pathway among human melanomas. Although combination therapy exerts significantly better antitumor cell efficacy, complete remission is rarely achieved. To employ an alternative approach, we have targeted the Hedgehog/GLI pathway, which is deregulated in melanomas, through the GLI1/2 inhibitor GANT61, alone or accompanied with the treatment by the BCL2 family inhibitor obatoclax in 9 melanoma cell lines. Thus, we targeted melanoma cells irrespective of their NRAS or BRAF mutational status. After GANT61 treatment, the cell viability was drastically diminished via apoptosis, as substantial nuclear DNA fragmentation was detected. In all tested melanoma cell lines, the combined treatment was more efficient than the application of each drug alone at the end of the cell growth with inhibitors. GANT61 was efficient also alone in most cell lines without the addition of obatoclax, which had only a limited effect when used as a single drug. In most cell lines, tumor cells were eradicated after 5-9 days of combined treatment in colony outgrowth assay. To conclude, GANT61 treatment might become a hopeful and effective anti-melanoma targeted therapy, especially when combined with the BCL2 family inhibitor obatoclax.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; GTP Phosphohydrolases; Humans; Indoles; Kruppel-Like Transcription Factors; Melanoma; Membrane Proteins; Mutation; Nuclear Proteins; Proto-Oncogene Proteins B-raf; Pyridines; Pyrimidines; Pyrroles; Zinc Finger Protein Gli2

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Biphenyl Compounds; Calcium; Cell Line, Tumor; Cytosol; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Humans; Indoles; Melanoma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Unfolded Protein Response; Up-Regulation

Constitutive activation of nuclear factor κB (NF-κB) characterizes melanoma cells. To explore the molecular mechanism of melanoma cell survival by constitutive NF-κB activation, we used the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), which directly binds to NF-κB. DHMEQ abrogated constitutive NF-κB activity, which included RelA (p65)/p50 in melanoma cell lines G361 and HMV-II; however, the reduction of the viability was marginal. Expression of c-FLIP was not observed in the melanoma cell lines tested, and DHMEQ could not repress the expression of the Bcl-2 family proteins Bcl-2 and Bcl-xL. Concomitant treatment with DHMEQ and the inhibitor of antiapoptotic Bcl-2 family proteins, GX15-070, triggered synergistic reduction of the viability and induced apoptosis of G361 cells. These results indicate that abrogation of the NF-κB pathway alone is not sufficient to suppress the survival of melanoma cells. The NF-κB and the antiapoptotic Bcl-2 pathways cooperatively support the survival, and the dual targeting triggers synergistic reduction of the viability and induces apoptosis of melanoma cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Cell Line, Tumor; Cell Survival; Cyclohexanones; Drug Synergism; Humans; Indoles; Melanoma; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Signal Transduction; Tumor Cells, Cultured

The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modeling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

Topics: Anti-Bacterial Agents; Antineoplastic Agents; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Indoles; Insulin Receptor Substrate Proteins; Mechanistic Target of Rapamycin Complex 1; Melanoma; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Prodigiosin; Proteins; Proto-Oncogene Proteins c-akt; Pyrroles; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases; Transcription Factors

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.

Topics: Antiviral Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Blotting, Western; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Immunoprecipitation; Indoles; Melanoma; Membrane Potential, Mitochondrial; Myeloid Cell Leukemia Sequence 1 Protein; Oxidative Stress; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; RNA, Small Interfering; Thapsigargin; Tumor Cells, Cultured; Tunicamycin

Topics: Antineoplastic Agents; Controlled Clinical Trials as Topic; Humans; Hydrazines; Indoles; Melanoma; Neoplasm Metastasis; Neoplasms; Pyrroles

Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Boronic Acids; Bortezomib; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Mice; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Pyrroles