pd-184352 and Melanoma

Phenotype-guided re-profiling of approved drug molecules presents an accelerated route to developing anticancer therapeutics by bypassing the target-identification bottleneck of target-based approaches and by sampling drugs already in the clinic. Further, combinations incorporating targeted therapies can be screened for both efficacy and toxicity. Previously we have developed an oncogenic-RAS-driven zebrafish melanoma model that we now describe display melanocyte hyperplasia while still embryos. Having devised a rapid method for quantifying melanocyte burden, we show that this phenotype can be chemically suppressed by incubating V12RAS transgenic embryos with potent and selective small molecule inhibitors of either MEK or PI3K/mTOR. Moreover, we demonstrate that combining MEK inhibitors (MEKi) with dual PI3K/mTOR inhibitors (PI3K/mTORi) resulted in a super-additive suppression of melanocyte hyperplasia. The robustness and simplicity of our novel screening assay inspired us to perform a modest screen of FDA approved compounds for their ability to potentiate MEKi PD184352 or PI3K/mTORi NVPBEZ235 suppression of V12RAS-driven melanocyte hyperplasia. Through this route, we confirmed Rapamycin as a compound that could synergize with MEKi and even more so with PI3K/mTORi to suppress melanoma development, including suppressing the growth of cultured human melanoma cells. Further, we discovered two additional compounds-Disulfiram and Tanshinone-that also co-operate with MEKi to suppress the growth of transformed zebrafish melanocytes and showed activity toward cultured human melanoma cells. In conclusion, we provide proof-of-concept that our phenotype-guided screen could be used to identify compounds that affect melanoma development and prompt further evaluation of Disulfiram and Tanshinone as possible partners for combination therapy.

Topics: Abietanes; Animals; Animals, Genetically Modified; Apoptosis; Benzamides; Cell Line, Tumor; Disease Models, Animal; Disulfiram; Drug Repositioning; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinases; Melanins; Melanocytes; Melanoma; Oligonucleotides, Antisense; Phenotype; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Skin Neoplasms; Zebrafish

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Caspases; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Enzyme Activation; Fibroblasts; Humans; Indoles; Melanoma; Mitogen-Activated Protein Kinases; Mutation; Oncogene Protein p21(ras); Oncolytic Virotherapy; Oncolytic Viruses; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reoviridae; Signal Transduction; Sulfonamides; Tumor Necrosis Factor-alpha; Xenograft Model Antitumor Assays

Inhibition of RAF/MEK/ERK signaling is beneficial for many patients with BRAF(V600E)-mutated melanoma. However, primary and secondary resistances restrict long-lasting therapy success. Combination therapies are therefore urgently needed. Here, we evaluate the cellular effect of combining a MEK inhibitor with a genotoxic apoptosis inducer. Strikingly, we observed that an activated MAPK pathway promotes in several melanoma cell lines the pro-apoptotic response to genotoxic stress, and MEK inhibition reduces intrinsic apoptosis. This goes along with MEK inhibitor induced increased RAS and P-AKT levels. The protective effect of the MEK inhibitor depends on PI3K signaling, which prevents the induction of pro-apoptotic PUMA that mediates apoptosis after DNA damage. We could show that the MEK inhibitor dependent feedback loop is enabled by several factors, including EGF receptor and members of the SPRED family. The simultaneous knockdown of SPRED1 and SPRED2 mimicked the effects of MEK inhibitor such as PUMA repression and protection from apoptosis. Our data demonstrate that MEK inhibition of BRAF(V600E)-positive melanoma cells can protect from genotoxic stress, thereby achieving the opposite of the intended anti-tumorigenic effect of the combination of MEK inhibitor with inducers of intrinsic apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Proliferation; Cisplatin; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Indazoles; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Melanoma; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation, Missense; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sulfonamides

The RAS/BRAF/MEK/ERK signaling pathway is a central driver in cancer with many BRAF and MEK inhibitors being evaluated in clinical trials. Identifying noninvasive biomarkers of early pharmacodynamic responses is important for development of these targeted drugs. As increased aerobic glycolysis is often observed in cancer, we hypothesized that MEK1/2 (MAP2K1/MAP2K2) inhibitors may reduce lactate levels as detected by magnetic resonance spectroscopy (MRS), as a metabolic biomarker for the pharmacodynamic response. MRS was used to monitor intracellular and extracellular levels of lactate in human cancer cells in vitro and in melanoma tumors ex vivo. In addition, we used (1)H MRS and a fluorescent glucose analog to evaluate the effect of MEK inhibition on glucose uptake. MEK1/2 signaling inhibition reduced extracellular lactate levels in BRAF-dependent cells but not BRAF-independent cells. The reduction in extracellular lactate in BRAF-driven melanoma cells was time-dependent and associated with reduced expression of hexokinase-II driven by c-Myc depletion. Taken together, these results reveal how MEK1/2 inhibition affects cancer cell metabolism in the context of BRAF oncogene addiction. Furthermore, they offer a preclinical proof-of-concept for the use of MRS to measure lactate as a noninvasive metabolic biomarker for pharmacodynamic response to MEK1/2 inhibition in BRAF-driven cancers.

Topics: Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Deoxyglucose; Diphenylamine; Female; Gene Knockdown Techniques; Glucose; Hexokinase; Humans; Lactic Acid; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Mice; Mutation, Missense; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Tumor Burden; Xenograft Model Antitumor Assays

Topics: Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Humans; Melanoma; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Ubiquitin-Protein Ligases

The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.. We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided.. Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).. Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Benzimidazoles; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Mice, Nude; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Paired Box Transcription Factors; PAX3 Transcription Factor; Real-Time Polymerase Chain Reaction; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays; Zebrafish

Resistance to BRAF(V600E) inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAF(V600E) inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF ( V600E ) or NRAS ( G12D ) and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAF(V600E) promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF ( V600E ) or NRAS ( G12D )-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAF(V600E)- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAF(V600E)- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 ( nu ) mice (P = .03 when mean tumor volume at day 13 was compared for BRAF(V600E) inhibitor vs BRAF(V600E) inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4-8 mice per group).

Topics: Animals; Antineoplastic Agents; Aspartic Acid; Benzamides; Blotting, Western; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p21; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, ras; Glutamic Acid; Glycine; Humans; MAP Kinase Signaling System; Melanocytes; Melanoma; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Transcription Factor AP-1; Transfection; Transplantation, Heterologous; Valine; Viral Vaccines

Most melanoma cells are characterized by the V600E mutation in B-Raf kinase. This mutation leads to increased expression of interleukin (CXCL8), which plays a key role in cell growth and angiogenesis. Thus CXCL8 appears to be an interesting therapeutic target. Hence, we performed vaccination of mice with GST-CXCL8, which results in a reduced incidence of syngenic B16 melanoma cell xenograft tumors. We next addressed the molecular mechanisms responsible for aberrant CXCL8 expression in melanoma. The CXCL8 mRNA contains multiples AU-rich sequences (AREs) that modulate mRNA stability through the binding of tristetraprolin (TTP). Melanoma cell lines express very low TTP levels. We therefore hypothesized that the very low endogenous levels of TTP present in different melanoma cell lines might be responsible for the relative stability of CXCL8 mRNAs. We show that TTP is actively degraded by the proteasome and that extracellular-regulated kinase inhibition results in TTP accumulation. Conditional expression of TTP in A375 melanoma cells leads to CXCL8 mRNA destabilization via its 3' untranslated regions (3'-UTR), and TTP overexpression reduces its production. In contrast, downregulation of TTP by short hairpin RNA results in upregulation of CXCL8 mRNA. Maintaining high TTP levels in melanoma cells decreases cell proliferation and autophagy and induces apoptosis. Sorafenib, a therapeutic agent targeting Raf kinases, decreases CXCL8 expression in melanoma cells through reexpression of TTP. We conclude that loss of TTP represents a key event in the establishment of melanomas through constitutive expression of CXCL8, which constitutes a potent therapeutic target.

Topics: Animals; Antibodies; Antineoplastic Agents; Apoptosis; Autophagy; Benzamides; Benzenesulfonates; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL1; Chemokine CXCL5; Dichlororibofuranosylbenzimidazole; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Genes, Reporter; Half-Life; Humans; Immunotherapy, Active; Interleukin-8; Leupeptins; MAP Kinase Kinase Kinases; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Niacinamide; Phenylurea Compounds; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins; Pyridines; Receptors, Interleukin-8B; RNA Stability; RNA, Messenger; RNA, Small Interfering; Sorafenib; Transfection; Tristetraprolin; Tumor Cells, Cultured; Vaccination

Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF have been identified in approximately 85% of melanomas, and components of this pathway have been developed as molecular targets for therapeutic intervention. We and others have shown that inhibition of this pathway with specific small molecule MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors induces a wide range of apoptotic responsiveness in human melanoma cells both in vitro and in vivo. To define the molecular mechanism underlying variable apoptotic sensitivity of melanoma cells to MEK inhibition, we examined the expression and subcellular localization of Bcl-2 family members in a comprehensive set of human melanoma cell lines. Whereas the proapoptotic protein Bim was activated and localized to the mitochondrial membrane in all cell lines regardless of apoptotic sensitivity, Bmf activation and cytosolic translocation was exclusive to sensitive cells. In resistant cells, Bmf remained sequestered to the cytoskeleton through dynein light chain 2 (DLC2) binding. Overexpression of Bmf in resistant cells did not enhance apoptosis, whereas expression of mutant BmfA69P, which has decreased binding to DLC2, promoted cell death. Expression of BmfA69P mutants possessing the Bcl-2 homology 3 (BH3) domain mutation L138A, which impairs BH3 interactions, did not enhance apoptosis in resistant cells. RNA interference targeting Bim and Bmf provided protection from apoptosis induced by MEK inhibition. These results show a novel role for Bmf in promoting apoptosis and provide insight into the mechanism of apoptotic resistance to MEK inhibition in melanoma.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Benzamides; Biphenyl Compounds; Caspases; Cell Line, Tumor; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Humans; Melanoma; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzamides; Butadienes; Cell Line, Tumor; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Humans; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Molecular Structure; Nitriles; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA Interference; Sulfones; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 micromol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the

Topics: Benzamides; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Dose-Response Relationship, Radiation; Enzyme Inhibitors; Humans; Leupeptins; MAP Kinase Signaling System; Melanoma; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Radiation-Sensitizing Agents; Time Factors; Transfection

The Ras-Raf-MAPK pathway is constitutively activated in the majority of melanomas because of a mutation in the BRAF gene. It has been hypothesized that activation of this pathway is crucial for the genesis and maintenance of melanoma and therefore represents an attractive clinical target for metastatic disease. We synthesized a previously characterized MAP kinase kinase inhibitor to test the effect that blocking the Ras-Raf-MAPK pathway would have on the establishment and maintenance of melanoma metastases. Oral administration of CI 1040 inhibited formation of pulmonary metastases and caused rapid regression of established pulmonary metastases in the mouse. Our findings indicate that Ras-Raf-MAPK activation provides crucial signals for the survival of melanoma cells at ectopic sites and that the pharmacological inhibition of this pathway is a promising target for melanoma therapy.

Topics: Animals; Benzamides; Cell Line, Tumor; Enzyme Inhibitors; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Melanoma; Mice; Mice, SCID; Mitogen-Activated Protein Kinases; Oncogene Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; ras Proteins; Xenograft Model Antitumor Assays