plx-4720 and Melanoma

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.

Topics: Clinical Trials as Topic; Humans; Melanoma; Molecular Weight; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Structure-Activity Relationship

While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly. One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis. We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination. The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome. We tested this hypothesis in cell lines and in mice. Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression. Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A. No significant changes were observed with BCL-2. Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID. No changes in mRNA or protein correlated with response. Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment. In xenograft models, navitoclax enhanced the efficacy of PLX4720. The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance. Trial registrations: ClinicalTrials.gov NCT01006980; ClinicalTrials.gov NCT01107418; ClinicalTrials.gov NCT01264380; ClinicalTrials.gov NCT01248936; ClinicalTrials.gov NCT00949702; ClinicalTrials.gov NCT01072175.

Topics: Adult; Aged; Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Imidazoles; Indoles; Melanoma; Mice; Middle Aged; Mutation, Missense; Neoplasm Metastasis; Oximes; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Pyridones; Pyrimidinones; Sulfonamides; Vemurafenib

The serine-threonine kinase, BRAF, is an upstream regulator of the MEK-ERK1/2 pathway and is commonly mutated in cancer. 14-3-3 proteins bind to two sites in BRAF, N-terminal S365, and C-terminal S729. 14-3-3 binding modulates the activity and dimerization of both wild-type and non-V600 mutant forms of BRAF. In BRAF V600E mutants, the C-terminal S729 site affects dimerization of truncated splice variants. The N-terminal, S365, is removed in BRAF V600E splice variants but its importance in full-length BRAF V600 mutants remains uncertain. We tested the role of S365 in dimerization and RAF inhibitor resistance in full-length BRAF V600E. Mutating BRAF S365 site to an alanine (S365A) reduced 14-3-3 association and increased BRAF V600E homodimerization. BRAF V600E S365A displayed reduced sensitivity to RAF inhibitor at the level of MEK-ERK1/2 signaling, cell growth, and cell viability. These data suggest that alteration or removal of the S365 14-3-3 binding site may contribute to RAF inhibitor resistance.

Topics: Binding Sites; Cell Line, Tumor; Humans; Indoles; Melanoma; Mutation; Protein Kinase Inhibitors; Protein Multimerization; Proto-Oncogene Proteins B-raf; Serine; Signal Transduction; Sulfonamides

Approximately 60% of patients with melanoma harbor BRAF mutation and targeting BRAF offers enormous advance in the treatment of those patients. Unfortunately, the efficacy of the BRAF inhibitors is usually restricted by the onset of drug resistance. Therefore, better understanding of the adaptive drug resistance mechanisms is essential for the development of alternative therapeutic strategies, and offers more promising measures to promote the short duration of response to BRAF inhibitors.. The levels of tumor suppressive long noncoding RNA on chromosome 8p12 (TSLNC8) were evaluated by qPCR. The MTT assay, colony formation assay, apoptosis assay, and in vivo xenograft tumor model were performed to assess the functions of TSLNC8 on drug resistance. Western blotting, RNA pull-down, and RNA immunoprecipitation (RIP) assays were applied to investigate the mechanisms of TSLNC8 in melanoma.. Herein, our findings demonstrate that TSLNC8 is significantly downregulated in BRAF inhibitor-resistant melanoma tissues and cells. Moreover, downregulation of TSLNC8 in BRAF inhibitor sensitive cells reduces the toxicity response to BRAF inhibitor PLX4720, and inhibits apoptosis of melanoma cells-treated with PLX4720. Further assay elucidates that TSLNC8 can bind with the catalytic subunit of protein phosphatase 1α (PP1α) to regulate its distribution, and Downregulation of TSLNC8 results in PP1α cytoplasmic accumulation, thus re-activating the MAPK signaling. Eventually, the overexpression of TSLNC8 in BRAF inhibitor PLX4720-resistant melanoma cells restores the sensitive to BRAF inhibitor.. Collectively, our research provides a compelling rationale for resistance to BRAF inhibitor in melanoma, and the patient might benefit from the combinatorial therapy of BRAF inhibitors and lncRNA TSLNC8.

Topics: Animals; Apoptosis; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Female; HEK293 Cells; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Kinase Inhibitors; Protein Phosphatase 1; Proto-Oncogene Proteins B-raf; RNA, Long Noncoding; Sulfonamides; Xenograft Model Antitumor Assays

Constitutive activation of STAT3 can play a vital role in the development of melanoma. STAT3-targeted therapeutics are reported to show efficacy in melanomas harboring the BRAFV600E mutant and also in vemurafenib-resistant melanomas. We designed and synthesized a series of substituted nitric oxide (NO)-releasing quinolone-1,2,4-triazole/oxime hybrids, hypothesizing that the introduction of a STAT3 binding scaffold would augment their cytotoxicity. All the hybrids tested showed a comparable level of in vitro NO production. 7b and 7c exhibited direct binding to the STAT3-SH domain with IC

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Melanoma; Molecular Docking Simulation; Molecular Structure; Nitric Oxide; Proto-Oncogene Proteins B-raf; STAT3 Transcription Factor; Structure-Activity Relationship

BRAF is a serine/threonine kinase that harbors activating mutations in ∼7% of human malignancies and ∼60% of melanomas. Despite initial clinical responses to BRAF inhibitors, patients frequently develop drug resistance. To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma, we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors. To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas, we integrate expression, ATAC-seq, and CRISPR screen data. We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6, which together enable resistance to BRAF inhibitors in melanoma cells. Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720, providing new insights into gene regulation in BRAF inhibitor resistant melanoma cells.

Topics: Cell Line, Tumor; Cell Proliferation; Clustered Regularly Interspaced Short Palindromic Repeats; Drug Resistance, Neoplasm; Humans; Indoles; Intercellular Signaling Peptides and Proteins; Melanoma; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides

Selective inhibition of the mutant BRAF protein is a highly promising therapeutic approach for melanoma patients carrying the BRAF mutation. Despite the remarkable clinical response, most patients develop resistance and experience tumour regrowth. To clarify the molecular background of BRAF inhibitor resistance, we generated four drug-resistant melanoma cell lines from paired primary/metastatic cell lines using a vemurafenib analogue PLX4720. Three of the resistant cell lines showed decreased proliferation after drug withdrawal, but the proliferation of one cell line (WM278) increased notably. Furthermore, we observed opposite phenomena in which a 'drug holiday' could not only be beneficial but also contribute to tumour progression. Using genomic and proteomic approaches, we found significantly different alterations between the sensitive and resistant cell lines, some of which have not been reported previously. In addition to several other changes, copy number gains were observed in all resistant cell lines on 8q24.11-q24.12 and 8q21.2. Gene expression analysis showed that most genes upregulated in the resistant cell lines were associated with cell motility and angiogenesis. Increased expression of six proteins (ANGPLT4, EGFR, Endoglin, FGF2, SerpinE1 and VCAM-1) and decreased expression of two proteins (osteopontin and survivin) were observed consistently in all resistant cell lines. In summary, we identified new genomic alterations and characterized the protein expression patterns associated with the resistant phenotype. Although several proteins have been shown to be associated with BRAF resistance, our study is the first to describe the association of VCAM-1 and osteopontin with BRAF resistance.

Topics: Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Molecular Targeted Therapy; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Skin Neoplasms; Sulfonamides; Survival Rate

Acquired resistance is a significant clinical challenge in targeted therapy of melanomas using BRAF inhibitors. We previously identified that downregulation of miR-92a-1-5p confers acquired resistance to BRAF inhibition using an miRNA array platform.. In this study, we investigated the target genes of miR-92a-1-5p and their functional significance in BRAF inhibitor resistance.. The miRNA target prediction data were combined with RNA-Seq data to identify possible target genes for miR-92a-1-5p. Cellular effects of target genes were further examined using siRNA knockdown, WST-1 assay, and immunoblotting analysis.. We selected S100 calcium-binding protein A9 (S100A9) as a possible target gene for functional validation. S100A9 knockdown abrogated resistance to PLX4720 in A375P/Mdr cells. This result was similar to those described earlier for miR-92a-1-5p, indicating that miR-92a-1-5p inhibits cell viability by targeting S100A9. S100A9 overexpression partially conferred PLX4720 resistance to A375P cells. We also demonstrated that MAPK re-activation does not contribute to the promotion of BRAF inhibitor resistance by S100A9.. Taken together, our results indicate that S100A9 might be functionally involved in development of resistance to BRAF inhibitors and might be a target for melanoma therapy in the future.

Topics: Antineoplastic Agents; Calgranulin B; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; MicroRNAs; Proto-Oncogene Proteins B-raf; Sulfonamides; Up-Regulation

Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Indoles; MAP Kinase Kinase Kinases; Melanoma; Mice; Mice, SCID; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridines; Sulfonamides; Xenograft Model Antitumor Assays

Many recent studies have uncovered the necessary role for the receptor-interacting protein kinase 1 (RIP1) in regulating apoptosis and necrosis that cells undergo in response to various cellular stresses. However, the functional significance of RIP1 in promoting cancer cells survival remains poorly understood. Here, we report that RIP1 was upregulated and contributed to both intrinsic and acquired resistance of melanoma cells to BRAF/MEK inhibitors through activation of NF-κB. Strikingly, Snail1-mediated suppression of CYLD played a crucial role in promoting RIP1 expression upon ERK activation, particularly, in melanoma cells with acquired resistance to BRAF inhibitors. In addition, RIP1 kinase activity was not required for melanoma cells to survive BRAF/MEK inhibition as RIP1 mediated NF-κB activation through its intermediate domain. Collectively, our findings reveal that targeting RIP1 in combination with BRAF/MEK inhibitors is a potential approach in the treatment of the disease.

Topics: Apoptosis; Cell Line, Tumor; Cytoprotection; Deubiquitinating Enzyme CYLD; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Mitogen-Activated Protein Kinase Kinases; Models, Biological; NF-kappa B; Nuclear Pore Complex Proteins; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; RNA-Binding Proteins; Snail Family Transcription Factors; Sulfonamides; Up-Regulation

Single-cell RNA-seq's (scRNA-seq) unprecedented cellular resolution at a genome-wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-seq data sets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present a

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sequence Analysis, RNA; Single-Cell Analysis; Sulfonamides

Drug resistance is the single most important driver of cancer treatment failure for modern targeted therapies, and the dialog between tumor and stroma has been shown to modulate the response to molecularly targeted therapies through proliferative and survival signaling. In this work, we investigate interactions between a growing tumor and its surrounding stroma and their role in facilitating the emergence of drug resistance. We used mathematical modeling as a theoretical framework to bridge between experimental models and scales, with the aim of separating intrinsic and extrinsic components of resistance in

Topics: Animals; Drug Resistance, Neoplasm; Focal Adhesion Kinase 1; Humans; Indoles; Melanoma; Mice; Models, Theoretical; Molecular Targeted Therapy; Proto-Oncogene Proteins B-raf; Stromal Cells; Sulfonamides; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

Microphthalmia-associated transcription factor (MITF) is expressed in melanomas and has a critical role in melanocyte development and transformation. Because inhibition of MITF inhibits cell growth in melanoma, MITF is a potential therapeutic target molecule. Here, we report the identification of CH6868398, which has a novel chemical structure and suppresses MITF expression at the protein level in melanoma cells. CH6868398 showed cell growth inhibition activity against MITF-dependent melanoma cells both with and without BRAF mutation and also exhibited anti-tumor efficacy in a melanoma xenograft model. Because selective BRAF inhibitors are standard therapeutics for BRAF-mutated melanoma, we investigated the effect of CH6868398 with a BRAF inhibitor, PLX4720, on cell growth inhibition. The addition of CH6868398 enhanced the cell growth inhibition activity of PLX4720 in melanoma cell lines. Furthermore, combination of CH6868398 and PLX4720 efficiently suppressed MITF protein and enhanced cleavage of Caspase3 and poly (ADP-ribose) polymerase (PARP) in melanoma cell lines. These data support the therapeutic potential of CH6868398 as an anti-melanoma agent that reduces MITF protein levels in combination with BRAF inhibitors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Female; Humans; Indoles; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Microphthalmia-Associated Transcription Factor; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Random Allocation; Sulfonamides; Xenograft Model Antitumor Assays

Treatment of BRAF mutant melanoma with kinase inhibitors has been associated with rapid tumor regression; however, this clinical benefit is short-lived, and most patients relapse. A number of studies suggest that the extracellular environment promotes BRAF inhibitor resistance and tumor progression. Extracellular vesicles, such as exosomes, are functional mediators in the extracellular environment. They are small vesicles known to carry a concentrated group of functional cargo and serve as intercellular communicators not only locally but also systemically. Increasingly, it is reported that extracellular vesicles facilitate the development of drug resistance in cancer; however, their role in BRAF inhibitor resistance in melanoma is unclear. Here we investigated if extracellular vesicles from BRAF inhibitor-resistant melanoma could influence drug sensitivity in recipient melanoma cells. We demonstrate that the resistance driver, PDGFRβ, can be transferred to recipient melanoma cells via extracellular vesicles, resulting in a dose-dependent activation of PI3K/AKT signaling and escape from MAPK pathway BRAF inhibition. These data suggest that the BRAF inhibitor-sensitive phenotype of metastatic melanoma can be altered by delivery of PDGFRβ by extracellular vesicles derived from neighboring drug-resistant melanoma cells.

Topics: Animals; Cattle; Cell Proliferation; Coculture Techniques; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Exosomes; Extracellular Fluid; Extracellular Vesicles; Humans; Indoles; Melanoma; Proto-Oncogene Proteins B-raf; Receptor, Platelet-Derived Growth Factor beta; Sulfonamides; Tumor Cells, Cultured

Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution.

Topics: Antineoplastic Agents, Hormonal; Benzimidazoles; Cell Line, Tumor; Estradiol; Evolution, Molecular; Female; Fulvestrant; Humans; Imidazoles; Indoles; Male; Melanoma; Oximes; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Pyridones; Pyrimidinones; Quinolines; Signal Transduction; Sulfonamides

Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation. Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome. We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity. We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis. Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism. The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines. Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics. Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Glucose; Glycolysis; Humans; Indoles; Melanoma; Molecular Targeted Therapy; Mutation; Oncogenes; Pharmacogenomic Variants; Phenotype; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Treatment Outcome

Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; B-Lymphocytes; Cancer Vaccines; Drug Antagonism; Female; gp100 Melanoma Antigen; Immunotherapy; Indoles; Melanoma; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Toll-Like Receptor 9; Tumor Cells, Cultured; Vaccines, Subunit

A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-Raf(V600E) inhibitors through scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the discovery of potent B-Raf(V600E) inhibitors with good oral bioavailability in rats and dogs. One of the compounds EBI-907 (13g) demonstrated excellent in vivo efficacy in B-Raf(V600E) dependent Colo-205 tumor xenograft models in mouse and is under preclinical studies for the treatment of melanoma and B-Raf(V600E) associated cancers.

Topics: Administration, Oral; Animals; Binding Sites; Cell Line, Tumor; Dogs; Drug Evaluation, Preclinical; Half-Life; Humans; Isoquinolines; Melanoma; Mice; Molecular Conformation; Molecular Dynamics Simulation; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyrazoles; Rats; Structure-Activity Relationship; Transplantation, Heterologous

Here, we retrospectively review imaging of 68 consecutive unselected patients with BRAF V600-mutant metastatic melanoma for organ-specific response and progression on vemurafenib. Complete or partial responses were less often seen in the central nervous system (CNS) (36%) and bone (16%) compared to lung (89%), subcutaneous (83%), spleen (71%), liver (85%) and lymph nodes/soft tissue (83%), P < 0.001. CNS was also the most common site of progression. Based on this, we tested in vitro the efficacy of the BRAF inhibitors PLX4720 and dabrafenib in the presence of cerebrospinal fluid (CSF). Exogenous CSF dramatically reduced cell death in response to both BRAF inhibitors. Effective cell killing was restored by co-administration of a PI-3 kinase inhibitor. We conclude that the efficacy of vemurafenib is variable in different organs with CNS being particularly prone to resistance. Extrinsic factors, such as ERK- and PI3K-activating factors in CSF, may mediate BRAF inhibitor resistance in the CNS.

Topics: Adolescent; Adult; Aged; Cell Death; Cell Survival; Central Nervous System Neoplasms; Disease Progression; Drug Resistance, Neoplasm; Female; Humans; Indoles; Male; Melanoma; Middle Aged; Organ Specificity; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Young Adult

Over half of BRAFV600E melanomas display intrinsic resistance to BRAF inhibitors, in part due to adaptive signaling responses. In this communication we ask whether BRAFV600E melanomas share common adaptive responses to BRAF inhibition that can provide clinically relevant targets for drug combinations. We screened a panel of 12 treatment-naïve BRAFV600E melanoma cell lines with MAP Kinase pathway inhibitors in pairwise combination with 58 signaling inhibitors, assaying for synergistic cytotoxicity. We found enormous diversity in the drug combinations that showed synergy, with no two cell lines having an identical profile. Although the 6 lines most resistant to BRAF inhibition showed synergistic benefit from combination with lapatinib, the signaling mechanisms by which this combination generated synergistic cytotoxicity differed between the cell lines. We conclude that adaptive responses to inhibition of the primary oncogenic driver (BRAFV600E) are determined not only by the primary oncogenic driver but also by diverse secondary genetic and epigenetic changes ("back-seat drivers") and hence optimal drug combinations will be variable. Because upregulation of receptor tyrosine kinases is a major source of drug resistance arising from diverse adaptive responses, we propose that inhibitors of these receptors may have substantial clinical utility in combination with inhibitors of the MAP Kinase pathway.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Humans; Indoles; Lapatinib; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Mice, SCID; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Quinazolines; Sulfonamides

Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two- and three-dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti-invasive potential of PI3K inhibitors and that drugs such as PX-866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E-BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Collagen; Gonanes; Indoles; Melanoma; Mice, Inbred NOD; Models, Biological; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular; Sulfonamides; Tumor Microenvironment

Treatment of BRAF-mutated melanoma tumors with BRAF inhibitor-based therapy produces high response rates, but of limited duration in the vast majority of patients. Published investigations of resistance mechanisms suggest numerous examples of tumor adaptation and signal transduction bypass mechanisms, but without insight into biomarkers that would predict which mechanism will predominate. Monitoring phenotypic response of multiple adaptive mechanisms simultaneously within the same tumor as it adapts during treatment has been elusive.. This study reports on a method to provide a more complete understanding of adaptive tumor responses. We simultaneously measured in vivo antitumor activity of 12 classes of inhibitors, which are suspected of enabling adaptive escape mechanisms, at various time points during systemic BRAF inhibition. We used implantable microdevices to release multiple compounds into distinct regions of a tumor to measure the efficacy of each compound independently and repeated these measurements as tumors progressed on systemic BRAF treatment.. We observed varying phenotypic responses to specific inhibitors before, during, and after prolonged systemic treatment with BRAF inhibitors. Our results specifically identify PI3K, PDGFR, EGFR, and HDAC inhibitors as becoming significantly more efficacious during systemic BRAF inhibition. The sensitivity to other targeted inhibitors remained mostly unchanged, whereas local incremental sensitivity to PLX4720 declined sharply.. These findings suggest redundancy of several resistance mechanisms and may help identify optimal constituents of more effective combination therapy in BRAF-mutant melanoma. They also represent a new paradigm for dynamic measurement of adaptive signaling mechanisms within the same tumor during therapy. Clin Cancer Res; 22(24); 6031-8. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Indoles; Melanoma; Mice; Mice, Nude; Mutation; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Xenograft Model Antitumor Assays

The majority of patients with melanoma harbor mutations in the BRAF oncogene, thus making it a clinically relevant target. However, response to mutant BRAF inhibitors (BRAFi) is relatively short-lived with progression-free survival of only 6 to 7 months. Previously, we reported high expression of ribonucleotide reductase M2 (RRM2), which is rate-limiting for de novo dNTP synthesis, as a poor prognostic factor in patients with mutant BRAF melanoma. In this study, the notion that targeting de novo dNTP synthesis through knockdown of RRM2 could prolong the response of melanoma cells to BRAFi was investigated. Knockdown of RRM2 in combination with the mutant BRAFi PLX4720 (an analog of the FDA-approved drug vemurafenib) inhibited melanoma cell proliferation to a greater extent than either treatment alone. This occurred in vitro in multiple mutant BRAF cell lines and in a novel patient-derived xenograft (PDX) model system. Mechanistically, the combination increased DNA damage accumulation, which correlated with a global decrease in DNA damage repair (DDR) gene expression and increased apoptotic markers. After discontinuing PLX4720 treatment, cells showed marked recurrence. However, knockdown of RRM2 attenuated this rebound growth both in vitro and in vivo, which correlated with maintenance of the senescence-associated cell-cycle arrest.. Inhibition of RRM2 converts the transient response of melanoma cells to BRAFi to a stable response and may be a novel combinatorial strategy to prolong therapeutic response of patients with melanoma. Mol Cancer Res; 14(9); 767-75. ©2016 AACR.

Topics: Animals; Cell Growth Processes; Cell Line, Tumor; Gene Knockdown Techniques; Humans; Indoles; Male; Melanoma; Mice; Molecular Targeted Therapy; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Random Allocation; Ribonucleoside Diphosphate Reductase; Sulfonamides; Xenograft Model Antitumor Assays

Therapeutic strategies for the treatment of metastatic melanoma show encouraging results in the clinic; however, not all patients respond equally and tumor resistance still poses a challenge. To identify novel therapeutic targets for melanoma, we screened a panel of structurally diverse organometallic inhibitors against human-derived normal and melanoma cells. We observed that a compound that targets PIM kinases (a family of Ser/Thr kinases) preferentially inhibited melanoma cell proliferation, invasion, and viability in adherent and three-dimensional (3D) melanoma models. Assessment of tumor tissue from melanoma patients showed that PIM kinases are expressed in pre- and post-treatment tumors, suggesting PIM kinases as promising targets in the clinic. Using knockdown studies, we showed that PIM1 contributes to melanoma cell proliferation and tumor growth in vivo; however, the presence of PIM2 and PIM3 could also influence the outcome. The inhibition of all PIM isoforms using SGI-1776 (a clinically-available PIM inhibitor) reduced melanoma proliferation and survival in preclinical models of melanoma. This was potentiated in the presence of the BRAF inhibitor PLX4720 and in the presence of PI3K inhibitors. Our findings suggest that PIM inhibitors provide promising additions to the targeted therapies available to melanoma patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Gene Expression Profiling; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Indoles; Melanoma; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; ortho-Aminobenzoates; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-pim-1; Pyridazines; Pyrimidinones; RNA Interference; Sulfonamides; Tumor Burden; Xenograft Model Antitumor Assays

Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents; Benzamides; Cell Line, Tumor; Cell Proliferation; Dioxoles; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Indoles; Melanocytes; Melanoma; Mice, Nude; Mitosis; Mutation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Skin Neoplasms; Smad4 Protein; Sulfonamides; Time Factors; Transfection; Transforming Growth Factor beta1; Vemurafenib; Xenograft Model Antitumor Assays; Zebrafish

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

Topics: Cell Line, Tumor; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA, Complementary; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Library; Genetic Engineering; Genetic Loci; Genetic Testing; Genome, Human; Humans; Indoles; Melanoma; Proto-Oncogene Proteins B-raf; Reproducibility of Results; RNA, Untranslated; Sulfonamides; Transcriptional Activation; Up-Regulation

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

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.

Topics: Animals; Animals, Genetically Modified; bcl-Associated Death Protein; Blotting, Southern; Blotting, Western; Cell Line, Tumor; Drug Resistance, Neoplasm; Embryonic Stem Cells; Exome; Genetic Association Studies; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Indoles; Melanoma; Mice; Mutagenesis; Oncogene Protein p21(ras); Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Signal Transduction; Sulfonamides; Transposases

Targeted inhibitors elicit heterogeneous clinical responses in genetically stratified groups of patients. Although most studies focus on tumor intrinsic properties, factors in the tumor microenvironment were recently found to modulate the response to inhibitors. Here, we show that in cutaneous BRAF V600E melanoma, the cytokine tumor necrosis factor-α (TNFα) blocks RAF inhibitor-induced apoptosis via activation of NF-κB. Several NF-κB-dependent factors are upregulated following TNFα and RAF inhibitor treatment. Of these factors, we show that death receptor inhibitor cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) is required for TNFα-induced protection against RAF inhibitor. Overexpression of c-FLIP_S or c-FLIP_L isoform decreased RAF inhibitor-induced apoptosis in the absence of TNFα. Importantly, targeting NF-κB enhances response to RAF inhibitor in vitro and in vivo. Together, our results show mechanistic evidence for cytokine-mediated resistance to RAF inhibitor and provide a preclinical rationale for the strategy of cotargeting the RAF/MEK/ERK1/2 pathway and the TNFα/NF-κB axis to treat mutant BRAF melanomas.

Topics: Animals; Apoptosis; Blotting, Western; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Indoles; Melanoma; Mice; Mice, Nude; NF-kappa B; Phosphatidylethanolamine Binding Protein; Proto-Oncogene Proteins B-raf; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Skin Neoplasms; Sulfonamides; Tumor Necrosis Factor-alpha

Intravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720. Initially, melanoma cells respond to PLX4720, but rapid reactivation of ERK/MAPK is observed in areas of high stromal density. This is linked to "paradoxical" activation of melanoma-associated fibroblasts by PLX4720 and the promotion of matrix production and remodeling leading to elevated integrin β1/FAK/Src signaling in melanoma cells. Fibronectin-rich matrices with 3-12 kPa elastic modulus are sufficient to provide PLX4720 tolerance. Co-inhibition of BRAF and FAK abolished ERK reactivation and led to more effective control of BRAF-mutant melanoma. We propose that paradoxically activated MAFs provide a "safe haven" for melanoma cells to tolerate BRAF inhibition.

Topics: Animals; Cell Line, Tumor; Drug Resistance, Neoplasm; Focal Adhesion Protein-Tyrosine Kinases; Humans; Indoles; Integrin beta1; Melanoma; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Tumor Microenvironment

The therapeutic efficacy of oncogenic BRAF inhibitor is limited by the onset of acquired resistance. In this study, we investigated the potential therapeutic effects of the mitotic inhibitor paclitaxel on three melanoma cell lines with differing sensitivity to the BRAF inhibitor. Of the two BRAF inhibitor-resistant cell lines, A375P/Mdr cells harboring the BRAF V600E mutant were resistant and the wild-type BRAF SK-MEL-2 cells were sensitive to paclitaxel. In particular, paclitaxel caused the growth inhibition of SK-MEL-2 cells to a much greater extent than it caused growth inhibition of A375P cells. Paclitaxel exhibited no significant effect on the phosphorylation of MEK-ERK in any cell lines tested, regardless of both the BRAF mutation and the drug resistance, implying that paclitaxel activity is independent of MEK-ERK inhibition. In A375P cells, paclitaxel treatment resulted in a marked emergence of apoptotic cells after mitotic arrest, concomitant with a remarkable induction of p21(Cip1). However, paclitaxel only moderately increased the levels of p21(Cip1) in A375P/Mdr cells, which exhibited a strong resistance to paclitaxel. The p21(Cip1) overexpression partially conferred paclitaxel sensitivity to A375P/Mdr cells. Interestingly, we found an extremely low background expression level of p21(Cip1) in SK-MEL-2 cells lacking normal p53 function, which caused much greater G2/M arrest than that seen in A375P cells. Taken together, these results suggest that paclitaxel may be an effective anticancer agent through regulating the expression of p21(Cip1) for the treatment of BRAF mutant melanoma cells resistant to BRAF inhibitors.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Mitosis; Mutation, Missense; Paclitaxel; Proto-Oncogene Proteins B-raf; Sulfonamides; Transcriptional Activation

Vertical growth phase (VGP) melanoma is frequently metastatic, a process mediated by changes in gene expression, which are directed by signal transduction pathways in the tumor cells. A prominent signaling pathway is the Ras-Raf-Mek-Erk MAPK pathway, which increases expression of genes that promote melanoma progression. Many melanomas harbor a mutation in this pathway, BRAF(V600E), which constitutively activates MAPK signaling and expression of downstream target genes that facilitate tumor progression. In BRAF(V600E) melanoma, the small molecule inhibitor, vemurafenib (PLX4032), has revolutionized therapy for melanoma by inducing rapid tumor regression. This compound down-regulates the expression of many genes. However, in this study, we document that blocking the Ras-Raf-Mek-Erk MAPK pathway, either with an ERK (PLX4032) or a MEK (U1026) signaling inhibitor, in BRAF(V600E) human and murine melanoma cell lines increases collagen synthesis in vitro and collagen deposition in vivo. Since TGFß signaling is a major mediator of collagen synthesis, we examined whether blocking TGFß signaling with a small molecule inhibitor would block this increase in collagen. However, there was minimal reduction in collagen synthesis in response to blocking TGFß signaling, suggesting additional mechanism(s), which may include activation of the p38 MAPK pathway. Presently, it is unclear whether this increased collagen synthesis and deposition in melanomas represent a therapeutic benefit or an unwanted "off target" effect of inhibiting the Ras-Raf-Erk-Mek pathway.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Collagen Type I; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Proto-Oncogene Proteins B-raf; raf Kinases; ras Proteins; Skin Neoplasms; Sulfonamides; Transforming Growth Factor beta; Vemurafenib; Xenograft Model Antitumor Assays

Mutations in the gene coding for the kinase B-Raf are associated with tumour growth in conjunctival melanoma. The purpose of this study is to explore effects of pharmacological B-Raf inhibition in conjunctival melanoma cell lines.. The B-Raf genotypes were assessed by PCR and subsequent sequencing. Cytotoxicity, cell viability, proliferation, apoptosis rate and phosphorylation rate of ERK and Akt were analysed in three different conjunctival melanoma cell lines under the influence of the B-Raf inhibitor PLX 4720 at various concentrations.. The cell lines CRMM-1 and CM2005.1 showed the B-Raf V600E mutation, whereas CRMM-2 expressed a B-Raf wild type. CM2005.1 was highly sensitive to PLX 4720, showing a complete cytotoxic effect for >1 µM, as well as a significant concentration-dependent reduction of the proliferation rate and viability rate. Even though CRMM-1 also carries the B-Raf V600E mutation, it did not react as sensitive to PLX 4720 inhibition as CM2005.1, but showed a significant concentration-dependent reduction regarding proliferation and viability. PLX 4720 had only slight impact on CRMM-2 in high concentrations (10 µM) regarding cytotoxicity, proliferation and viability. Fluorescence-activated cell sorting analysis revealed that PLX 4720 acted predominantly antiproliferative and not via an induction of apoptosis. The phosphorylation rate of ERK was significantly reduced in CRMM-1 and CM2005.1, while it remained unchanged in CRMM-2. The phosphorylation rate of Akt was significantly elevated in CRMM-2.. Proliferation inhibition of conjunctival melanoma cells by PLX 4720 depends on their B-Raf genotype. Therefore, therapeutic application of B-Raf inhibitors should take into account the specific B-Raf genotype.

Topics: Apoptosis; Cell Proliferation; Conjunctival Neoplasms; Enzyme-Linked Immunosorbent Assay; Genotype; Humans; Immunoblotting; Indoles; Melanoma; Polymerase Chain Reaction; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Tumor Cells, Cultured

Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.

Topics: Amino Acid Substitution; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Mutation, Missense; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins B-raf; Sulfonamides

A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B; Cell Death; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; High-Throughput Screening Assays; Humans; Indoles; Melanoma; Mice; Molecular Targeted Therapy; Proto-Oncogene Proteins B-raf; Quinazolines; Receptors, Platelet-Derived Growth Factor; Receptors, Vascular Endothelial Growth Factor; Sulfonamides; Xenograft Model Antitumor Assays

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAF(V600E) or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAF(V600E) into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma.

Topics: Activating Transcription Factor 6; Adaptation, Physiological; Apoptosis; Cell Line, Tumor; Cell Survival; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Heat-Shock Proteins; Humans; Indoles; MAP Kinase Signaling System; Melanocytes; Melanoma; Membrane Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Regulatory Factor X Transcription Factors; Skin Neoplasms; Sulfonamides; Transcription Factors; X-Box Binding Protein 1

Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors.. BRAF is suppress glycolysis and provide strong clinical benefi t in BRAF V600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors.

Topics: Cell Line, Tumor; Drug Resistance, Neoplasm; Glycolysis; HEK293 Cells; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridines; Sulfonamides; Transcription Factors; Vemurafenib

Resistance to RAF inhibitors such as vemurafenib and dabrafenib is a major clinical problem in the treatment of melanoma. Patients with mutant BRAF melanoma that progress on RAF inhibitors have limited treatment options, and drug removal from resistant tumors may elicit multiple effects. A frequent mechanism of resistance to RAF inhibitors is caused by expression of mutant BRAF splice variants. RAF inhibitor-resistant cell lines, generated in vivo, were tested as to whether or not mutant BRAF splice variants confer a fitness advantage in the presence of RAF inhibitor. Critically, cells expressing distinct mutant BRAF splice variants grow more efficiently in vitro and in vivo in the presence of the vemurafenib analog, PLX4720, compared with in the absence of inhibitor. PLX4720-treated BRAF splice variant-expressing cells exhibited levels of phospho-extracellular signal-regulated kinase (ERK)1/2 comparable to untreated parental cells. In addition, a reduction in phospho-ERK1/2 levels following treatment with the MEK inhibitor, trametinib (GSK1120212) phenocopied the fitness benefit provided by PLX4720. These data indicate that mutant BRAF splice variant-expressing melanoma cells are benefited by defined concentrations of RAF inhibitors.. This study provides evidence that RAF inhibitor-resistant melanoma cells benefit from continued therapy.

Topics: Animals; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Indoles; Melanoma; Mice; Mice, Nude; Protein Isoforms; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Sulfonamides; Xenograft Model Antitumor Assays

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

Topics: Animals; Antineoplastic Agents; Autophagy; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Mutation, Missense; Proto-Oncogene Proteins B-raf; Sulfonamides; Vemurafenib; Xenograft Model Antitumor Assays

There is currently no curative treatment for melanoma once the disease spreads beyond the original site. Although activation of the PI3K/Akt pathway resulting from genetic mutations and epigenetic deregulation of its major regulators is known to cause resistance of melanoma to therapeutic agents, including the conventional chemotherapeutic drug dacarbazine and the Food and Drug Administration-approved mutant BRAF inhibitors vemurafenib and dabrafenib, the role of extracellular stimuli of the pathway, such as insulin, in drug resistance of melanoma remains less understood.. To investigate the effect of insulin on the response of melanoma cells to dacarbazine, and in particular, the effect of insulin on the response of melanoma cells carrying the BRAF(V600E) mutation to mutant BRAF inhibitors. An additional aim was to define the role of the PI3K/Akt pathway in the insulin-triggered drug resistance.. The effect of insulin on cytotoxicity induced by dacarbazine or the mutant BRAF inhibitor PLX4720 was tested by pre-incubation of melanoma cells with insulin. Cytotoxicity was determined by the MTS assay. The role of the PI3K/Akt pathway in the insulin-triggered drug resistance was examined using the PI3K inhibitor LY294002 and the PI3K and mammalian target of rapamycin dual inhibitor BEZ-235. Activation of the PI3K/Akt pathway was monitored by Western blot analysis of phosphorylated levels of Akt.. Recombinant insulin attenuated dacarbazine-induced cytotoxicity in both wild-type BRAF and BRAF(V600E) melanoma cells, whereas it also reduced killing of BRAF(V600E) melanoma cells by PLX4720. Nevertheless, the protective effect of insulin was abolished by the PI3K and mTOR dual inhibitor BEZ-235 or the PI3K inhibitor LY294002.. Insulin attenuates the therapeutic efficacy of dacarbazine and PLX4720 in melanoma cells, which is mediated by activation of the PI3K/Akt pathway and can be overcome by PI3K inhibitors.

Topics: Animals; Cell Line, Tumor; Dacarbazine; Drug Resistance, Neoplasm; Humans; Indoles; Insulin; Melanoma; Mice; Mutation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Signal Transduction; Sulfonamides

Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF-ERK (extracellular signal-regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)-ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor-resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

Topics: Animals; Blotting, Western; Cell Shape; Dimethyl Sulfoxide; Drug Resistance, Neoplasm; Enzyme-Linked Immunosorbent Assay; Humans; Indoles; Interleukin-8; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Skin Neoplasms; Statistics, Nonparametric; Sulfonamides

The clinically approved oncogenic BRAF inhibitor PLX4032 (vemurafenib) was shown to be a substrate of the ATP-binding cassette (ABC) transporter ABCB1. Here, we compared PLX4032 and its structurally closely related precursor compound PLX4720 for their interference with ABCB1 and the ABCB1-mediated compound transport using docking and cell culture experiments.. For the docking study of PLX4032 and PLX4720 with ABCB1, we analysed binding of both compounds to mouse Abcb1a and to human ABCB1 using a homology model of human ABCB1 based on the 3D structure of Abcb1a. Naturally ABCB1 expressing cells including V600E BRAF-mutated and BRAF wild-type melanoma cells and cells transduced with a lentiviral vector encoding for ABCB1 were used as cell culture models. ABCB1 expression and function were studied by the use of fluorescent and cytotoxic ABCB1 substrates in combination with ABCB1 inhibitors.. Docking experiments predicted PLX4032 to interact stronger with ABCB1 than PLX4720. Experimental studies using different cellular models and structurally different ABCB1 substrates confirmed that PLX4032 interfered stronger with ABCB1 function than PLX4720. For example, PLX4032 (20 µM) induced a 4-fold enhanced rhodamine 123 accumulation compared to PLX4720 (20 µM) in ABCB1-transduced UKF-NB-3 cells and reduced the IC₅₀ for the cytotoxic ABCB1 substrate vincristine in this model by 21-fold in contrast to a 9-fold decrease induced by PLX4720.. PLX4032 exerted stronger effects on ABCB1-mediated drug transport than PLX4720. This indicates that small changes in a molecule can substantially modify its interaction with ABCB1, a promiscuous transporter that transports structurally different compounds.

Topics: Adenosine Triphosphatases; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; Cell Line; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Therapy, Combination; Flow Cytometry; Humans; Indoles; Melanoma; Mice; Molecular Docking Simulation; Proto-Oncogene Proteins B-raf; Sulfonamides; Vemurafenib; Vincristine

Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma.. Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.

Topics: Anilides; Benzimidazoles; Benzocycloheptenes; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Indoles; MAP Kinase Signaling System; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; NF-kappa B p50 Subunit; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Pyridines; Quinolines; Sulfonamides; Triazoles

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.

Topics: beta Catenin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Frizzled Receptors; Humans; Indoles; Melanoma; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; RNA Interference; Signal Transduction; Sulfonamides; Up-Regulation; Wnt Proteins; Wnt Signaling Pathway; Wnt-5a Protein

ERBB3/HER3 expression and signaling are upregulated in mutant BRAF melanoma as an adaptive, prosurvival response to FDA-approved RAF inhibitors. Because compensatory ERBB3 signaling counteracts the effects of RAF inhibitors, cotargeting ERBB3 may increase the efficacy of RAF inhibitors in mutant BRAF models of melanoma. Here, we corroborate this concept by showing that the ERBB3 function-blocking monoclonal antibody huHER3-8 can inhibit neuregulin-1 activation of ERBB3 and downstream signaling in RAF-inhibited melanoma cells. Targeting mutant BRAF in combination with huHER3-8 decreased cell proliferation and increased cell death in vitro, and decreased tumor burden in vivo, compared with targeting either mutant BRAF or ERBB3 alone. Furthermore, the likelihood of a durable tumor response in vivo was increased when huHER3-8 was combined with RAF inhibitor PLX4720. Together, these results offer a preclinical proof of concept for the application of ERBB3-neutralizing antibodies to enhance the efficacy of RAF inhibitors in melanoma to delay or prevent tumor regrowth. As ERBB3 is often upregulated in response to other kinase-targeted therapeutics, these findings may have implications for other cancers as well.

Topics: Animals; Antibodies, Monoclonal; Cell Growth Processes; Cell Line, Tumor; Cell Survival; Female; Heterografts; Humans; Indoles; Melanoma; Mice; Mice, Nude; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Receptor, ErbB-3; Signal Transduction; Sulfonamides; Vemurafenib

The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.

Topics: Animals; Blotting, Western; Catechin; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; DNA-Binding Proteins; Drug Resistance, Neoplasm; Enzyme Activation; Female; Histone Chaperones; Humans; Indoles; Melanoma; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Microscopy, Confocal; Mutation; Neurofibromin 2; Protein Phosphatase 2; Proto-Oncogene Proteins B-raf; Receptors, Laminin; RNA Interference; Sulfonamides; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

Various kinase inhibitors are known to be ATP-binding cassette (ABC) transporter substrates and resistance acquisition to kinase inhibitors has been associated to increased ABC transporter expression. Here, we investigated the role of the ABC transporters ABCB1, ABCC1, and ABCG2 during melanoma cell resistance acquisition to the V600-mutant BRAF inhibitors PLX4032 (vemurafenib) and PLX4720. PLX4032 had previously been shown to interfere with ABCB1 and ABCG2. PLX4720 had been demonstrated to interact with ABCB1 but to a lower extent than PLX4032.. PLX4032 and PLX4720 affected ABCC1- and ABCG2-mediated drug transport in a similar fashion. In a panel of 16 V600E BRAF-mutated melanoma cell lines consisting of four parental cell lines and their sub-lines with acquired resistance to PLX4032, PLX4720, vincristine (cytotoxic ABCB1 and ABCC1 substrate), or mitoxantrone (cytotoxic ABCG2 substrate), we detected enhanced ABC transporter expression in 4/4 cytotoxic ABC transporter substrate-resistant, 3/4 PLX4720-resistant, and 1/4 PLX4032-resistant melanoma cell lines.. PLX4032 has the potential to induce ABC transporter expression but this potential is lower than that of PLX4720 or cytotoxic ABC transporter substrates. Since ABC transporters confer multi-drug resistance, this is of relevance for the design of next-line therapies.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Multidrug Resistance-Associated Proteins; Mutation; Neoplasm Proteins; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Sulfonamides; Time Factors; Vemurafenib

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.

Topics: Cell Line, Tumor; Chromatography, Liquid; Down-Regulation; Forkhead Transcription Factors; Humans; Indazoles; Indoles; Melanoma; Molecular Targeted Therapy; Mutation; Piperazines; Proteomics; Proto-Oncogene Proteins B-raf; Receptor, ErbB-3; rho-Associated Kinases; RNA Interference; RNA, Small Interfering; Sulfonamides; Tandem Mass Spectrometry; Vemurafenib

The BRAF mutant, BRAF(V600E), is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF(V600E) metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF(V600E)-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf(V600E)-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf(V600E) mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8(+) T/FoxP3(+)CD4(+) T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemokine CCL2; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Humans; Immunity, Cellular; Immunotherapy; Indoles; Male; Melanoma; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Mutation, Missense; Proto-Oncogene Proteins B-raf; Receptors, CCR2; Sulfonamides; T-Lymphocytes, Regulatory; Tumor Necrosis Factor Receptor Superfamily, Member 9; Xenograft Model Antitumor Assays

Activating mutations in BRAF are the most common genetic alterations in melanoma. Inhibition of BRAF by small molecules leads to cell-cycle arrest and apoptosis. We show here that BRAF inhibition also induces an oxidative phosphorylation gene program, mitochondrial biogenesis, and the increased expression of the mitochondrial master regulator, PGC1α. We further show that a target of BRAF, the melanocyte lineage factor MITF, directly regulates the expression of PGC1α. Melanomas with activation of the BRAF/MAPK pathway have suppressed levels of MITF and PGC1α and decreased oxidative metabolism. Conversely, treatment of BRAF-mutated melanomas with BRAF inhibitors renders them addicted to oxidative phosphorylation. Our data thus identify an adaptive metabolic program that limits the efficacy of BRAF inhibitors.

Topics: Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Indoles; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Mitochondria; Mitogen-Activated Protein Kinases; Mutation; Oxidative Phosphorylation; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Proto-Oncogene Proteins B-raf; RNA, Messenger; Signal Transduction; Sulfonamides; Transcription Factors; Vemurafenib

The RAF inhibitor vemurafenib achieves remarkable clinical responses in mutant BRAF melanoma patients. However, vemurafenib is burdened by acquired drug resistance and by the side effects associated with its paradoxical activation of the ERK1/2 pathway in wild-type BRAF cells. This paradoxical effect has driven the development of a new class of RAF inhibitors. Here, we tested one of these selective, non-paradox-inducing RAF inhibitors termed paradox-breaker-04 (PB04) or PLX7904. Consistent with its design, PB04 is able to efficiently inhibit activation of ERK1/2 in mutant BRAF melanoma cells but does not hyperactivate ERK1/2 in mutant RAS-expressing cells. Importantly, PB04 inhibited ERK1/2 phosphorylation in mutant BRAF melanoma cells with acquired resistance to vemurafenib/PLX4720 that is mediated by a secondary mutation in NRAS. Consistent with ERK1/2 reactivation driving the re-acquisition of malignant properties, PB04 promoted apoptosis and inhibited entry into S phase and anchorage-independent growth in mutant N-RAS-mediated vemurafenib-resistant cells. These data indicate that paradox-breaker RAF inhibitors may be clinically effective as a second-line option in a cohort of acquired vemurafenib-resistant patients.

Topics: Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Line, Tumor; Collagen; Disease Progression; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Genes, ras; Humans; Indoles; Melanoma; Mutation; Phosphorylation; raf Kinases; ras Proteins; Sulfonamides; Vemurafenib

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors.

Topics: Cell Survival; Enzyme Inhibitors; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Indoles; Lapatinib; MAP Kinase Kinase Kinases; Melanoma; Oligonucleotide Array Sequence Analysis; Phosphorylation; Quinazolines; raf Kinases; Receptor, ErbB-3; Signal Transduction; Skin Neoplasms; Sulfonamides; Transcription, Genetic

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF(V600E) melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF(V600E) melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF(V600E) melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Drug Synergism; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Male; Melanoma; Mice; Mice, Nude; Mutation, Missense; Necrosis; Panobinostat; Proto-Oncogene Proteins B-raf; Receptor-Interacting Protein Serine-Threonine Kinases; RNA, Small Interfering; Sulfonamides; Vemurafenib; Vorinostat; Xenograft Model Antitumor Assays

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

Topics: Analysis of Variance; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Proliferation; DNA-Binding Proteins; Immunohistochemistry; Indoles; Jumonji Domain-Containing Histone Demethylases; Melanoma; Mice; Mutation, Missense; Phenformin; Proto-Oncogene Proteins B-raf; Sulfonamides

Melanoma antigen gene family (MAGE)-A4, a member of the cancer testis antigen family, has been reported in various cancers including melanoma, bladder, head and neck, oral, and lung, and is a potential target for T-cell-receptor-based immunotherapy. Baseline expression levels of the MAGE-A4 gene in thyroid cancer cell lines have not been previously studied thoroughly.. Human thyroid cancer cell lines (8505c, HTh7, BCPAP, and TPC-1) were treated with either 10 μmol/L 5'-azacytidine (Aza) or 10 μmol/L 5-AZA-2'deoxycytidine (DAC) and evaluated for various MAGEA gene expression. Later melanoma cell lines A375 and 8505c were treated with PLX4720 in combination with DAC and evaluated for MAGE-A4 expression.. Only BCPAP cells expressed moderate levels of MAGE-A3 and MAGE-A6 at baseline. Treatment with DAC/Aza induced the expression of MAGE-A4 and MAGE-A1 in 8505c cells. PLX4720 treatment did not affect MAGE-A4 expression in 8505c cells, but increased its expression in A375 cells. In contrast, addition of PLX4720 to DAC-treated 8505c cells decreased the previously induced MAGE-A4 expression by DAC in these cells. A similar decrease in MAGE-A4 expression by DAC was also seen in 8505cBRAF(-/-) cells. Although DAC treatment resulted in demethylation of the MAGE-A4 promoter in 2 CpG sites, PLX addition to DAC did not affect the demethylation status.. Demethylating agents increased the expression of MAGE genes in thyroid cancer cells. The effect of BRAFV600E inhibitors on MAGE-A4 expression suggest the role of downstream MEK/BRAF signaling in its expression apart from promoter demethylation being the sole requirement. Expression of MAGE-A4 may make immunotherapeutic intervention possible in selected patients with thyroid cancer.

Topics: Antigens, Neoplasm; Azacitidine; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Gene Expression; Humans; Immunotherapy; Indoles; MAP Kinase Signaling System; Melanoma; Neoplasm Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins B-raf; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms

Topics: 9,10-Dimethyl-1,2-benzanthracene; Administration, Topical; Animals; Antineoplastic Agents; Carcinogens; Carcinoma, Squamous Cell; Cell Line; Cell Proliferation; Fluorouracil; Humans; Imidazoles; Indoles; Melanoma; Mice; Oximes; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Sulfonamides; Tetradecanoylphorbol Acetate; Vemurafenib

Although the introduction of selective v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors has been a major advance in treatment of metastatic melanoma, approximately 50% of patients have limited responses including stabilization of disease or no response at all. This study aims to identify a novel means of overcoming resistance of melanoma to killing by BRAF inhibitors. We examined the influence of the BH3-mimetic ABT-737 on induction of apoptosis by the selective BRAF inhibitor PLX4720 in melanoma cells with or without BRAF V600E mutation. Included were cell lines established from four patients before and during treatment with selective BRAF inhibitors and 3D spheroids derived from these cell lines. Cell lines with no or low sensitivity to PLX4720 underwent synergistic increases and increased rates of apoptosis when combined with ABT-737. This degree of synergism was not seen in cell lines without BRAF V600E mutations. Apoptosis was mediated through the mitochondrial pathway and was due in part to upregulation of Bim as shown by inhibition of apoptosis following small interfering RNA knockdown of Bim. Similar effects were seen in cell lines established from patients prior to treatment but not in lines from patients clinically resistant to the selective BRAF inhibitors and in 3D spheroids derived from these cell lines. These results suggest that combination of selective BRAF inhibitors with ABT-737 or the related orally available compound ABT-263 may increase the degree and rate of responses in previously untreated patients with V600E melanoma but not in those with acquired resistance to these agents.

Topics: Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Proliferation; Clinical Trials, Phase II as Topic; Drug Resistance, Neoplasm; Drug Synergism; Humans; Immunoprecipitation; Indoles; Melanoma; Membrane Potential, Mitochondrial; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sulfonamides; Tumor Cells, Cultured

Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors.. BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2D(b) to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples.. We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model.. These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell-based immunotherapy for the treatment of patients with melanoma.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy, Adoptive; Indoles; Lymphocytes, Tumor-Infiltrating; Melanoma; Mice; Mutation; Promoter Regions, Genetic; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Sulfonamides; T-Lymphocytes; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Discovery of novel kinase inhibitors has matured rapidly over the last decade. Paramount to the successful development of kinase inhibitors is appropriate selectivity for validated targets. Many different approaches have been applied over the years, with varied results. There are currently thirteen different small molecule protein kinase inhibitors on the marketplace. Interestingly, a majority of these compounds lack precise selectivity for specific targets. This will change in the coming years, as technology for achieving improved selectivity becomes more widely applied. This chapter will focus on some of the critical considerations in setting up a kinase discovery and development project, citing examples particularly targeting the Raf kinases.

Topics: Adenosine Triphosphate; Animals; Crystallography, X-Ray; Drug Discovery; Drug Evaluation, Preclinical; Drug-Related Side Effects and Adverse Reactions; Enzyme Activation; Enzyme Assays; Humans; Indoles; Melanoma; Mice; Niacinamide; Phenylurea Compounds; Phosphorylation; Protein Kinase Inhibitors; raf Kinases; Solubility; Sorafenib; Structure-Activity Relationship; Sulfonamides

Melanoma cells driven by mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) are highly resistant to chemotherapeutic treatments. Recent phase 1 results with PLX4032/RG7204/vemurafenib, which selectively inhibits B-RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field. Nearly all patients in the phase 1-3 studies saw at least some response and the overall response rates ranged from 48 and 81%. However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time. These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032. It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond. We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is upregulated following inhibition of B-RAF-MEK signaling in mutant B-RAF melanoma cells. Here, we show that upregulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death. Small interfering RNA-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines. Additionally, upregulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death. Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment. Together, these data indicate that upregulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance.

Topics: Apoptosis; Cell Line, Tumor; Drug Resistance, Neoplasm; Forkhead Transcription Factors; Humans; Indoles; Melanoma; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Vemurafenib

To evaluate the effects of treatment with the potent mutant BRAF inhibitors GSK2118436 or vemurafenib (PLX4720) on immune responses to metastatic melanoma in tissues taken before and after treatment.. Thirty-seven tumor biopsies were collected from 15 patients with unresectable American Joint Committee on Cancer stage III or IV melanoma immediately before and approximately 7 days after the commencement of BRAF inhibitor treatment and at the time of tumor progression. Immunohistochemical staining was carried out on the biopsies using specific antibodies for CD8, CD4, CD20, CD1a, and Granzyme B.. Tumor infiltration by CD4(+) and CD8(+) lymphocytes increased markedly following BRAF inhibitor treatment (both ρ = 0.015). There was a correlation between the degree of tumor infiltration by CD8(+) and Granzyme B-expressing lymphocytes in post-BRAF inhibitor-treated biopsies (r = 0.690 and ρ = 0.013). Increased intratumoral CD8(+) lymphocyte expression was correlated with a reduction in tumor size and an increase in necrosis in posttreatment biopsies (r = -0.793, ρ = 0.011; and r = 0.761, ρ = 0.004, respectively).. The increase in tumor-infiltrating lymphocytes induced by treatment with BRAF inhibitors provides strong support for conducting trials that combine BRAF inhibitors with immunotherapy in the hope of prolonging clinical responses.

Topics: Adult; Aged; Female; Granzymes; Humans; Imidazoles; Indoles; Lymphocytes, Tumor-Infiltrating; Male; Melanoma; Middle Aged; Mutation; Neoplasm Metastasis; Neoplasm Staging; Oximes; Prognosis; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; T-Lymphocytes; Young Adult

BRAF inhibition is highly active in BRAF-mutant melanoma, but the degree and duration of responses is quite variable. Improved understanding of the mechanisms of de novo resistance may lead to rational therapeutic strategies with improved efficacy. Proteomic analysis of BRAF-mutant, PTEN-wild-type human melanoma cell lines treated with PLX4720 demonstrated that sensitive and de novo resistant lines exhibit similar RAS-RAF-MEK-ERK pathway inhibition, but the resistant cells exhibited durable activation of S6 and P70S6K. Treatment with the mTOR inhibitor rapamycin blocked activation of P70S6K and S6, but it also increased activation of AKT and failed to induce cell death. Combined treatment with rapamycin and PX-866, a PI3K inhibitor, blocked the activation of S6 and AKT and resulted in marked cell death when combined with PLX4720. The results support the rationale for combined targeting of BRAF and the PI3K-AKT pathways and illustrate how target selection will be critical to such strategies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Extracellular Signal-Regulated MAP Kinases; Gonanes; Humans; Indoles; Melanoma; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Interaction Maps; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Relatively little attention has been paid to the activity of selective BRAF inhibitors in the induction of apoptosis in melanoma, particularly in vivo. In the present study, we have isolated cultures from biopsies taken from four patients before and during the treatment of their melanoma. We report that the cell lines taken during treatment show varying degrees of upregulation of the proapoptotic BH3 protein Bim and its splice forms, downregulation of Mcl-1, and upregulation of the splicing factor SRp55 as reported in previous in-vitro studies. There was also evidence of ongoing apoptotic signaling despite the continued growth of the cultures. The cultures established during the treatment were largely resistant to the selective BRAF inhibitor PLX4720, consistent with the acquired resistance of melanoma in the treated patients. These results provide further insights into the mechanism of action of these agents against melanoma.

Topics: Adult; Aged; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biopsy; Cell Proliferation; Cell Shape; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Imidazoles; Indoles; Male; Melanoma; Membrane Proteins; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Nuclear Proteins; Oximes; Phosphoproteins; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA-Binding Proteins; Serine-Arginine Splicing Factors; Skin Neoplasms; Sulfonamides; Time Factors; Treatment Outcome; Tumor Cells, Cultured; Up-Regulation

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Coculture Techniques; Drug Resistance, Neoplasm; Hepatocyte Growth Factor; Humans; Indoles; Melanoma; Molecular Targeted Therapy; Mutation; Phosphatidylinositol 3-Kinases; Prognosis; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Signal Transduction; Stromal Cells; Sulfonamides; Tumor Microenvironment; Vemurafenib

The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.

Topics: Amino Acid Substitution; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Transformation, Neoplastic; Disease Models, Animal; Drug Administration Routes; Humans; Indoles; Integrases; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; Melanoma; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Mutation; Proto-Oncogene Proteins B-raf; PTEN Phosphohydrolase; Skin Neoplasms; Sulfonamides; Time Factors

The limitations of revolutionary new mutation-specific inhibitors of BRAF(V600E) include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF(V600E) by PLX4720 synergistically induces apoptosis across a spectrum of BRAF(V600E) melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.

Topics: Antineoplastic Agents; Apoptosis; Axin Protein; Benzimidazoles; beta Catenin; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Indoles; Melanoma; Mutation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; Signal Transduction; Sulfonamides; Wnt3A Protein

People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation; Hair Color; Indoles; Melanins; Melanoma; Mice; Mice, Inbred C57BL; Monophenol Monooxygenase; Peroxidases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Receptor, Melanocortin, Type 1; Skin Pigmentation; Sulfonamides; Survival Analysis; Tumor Cells, Cultured; Ultraviolet Rays

This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.. A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.. Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.. Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.

Topics: Animals; Antineoplastic Agents; Automation, Laboratory; Cell Line, Tumor; Drug Screening Assays, Antitumor; Female; Humans; Indoles; Inhibitory Concentration 50; MAP Kinase Signaling System; Melanoma; Mice; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Proto-Oncogene Proteins B-raf; Sulfonamides; Xenograft Model Antitumor Assays

Activating mutations in B-RAF and N-RAS occur in ∼60 and ∼15% of melanomas, respectively. The most common mutation in B-RAF is V600E, which activates B-RAF and the downstream MEK-ERK1/2 pathway. Thus, B-RAF(V600E) is a viable therapeutic target. PLX4720 is a selective inhibitor of mutant B-RAF and its analog, PLX4032, is currently undergoing clinical trials in melanoma. However, the effects of PLX4720 across the genotypic spectrum in melanoma remain unclear. Here, we describe that PLX4720 treatment rapidly induces hyperactivation of the MEK-ERK1/2 pathway in mutant N-RAS melanoma cells. Furthermore, we demonstrate that C-RAF is the major RAF isoform involved in this process. Importantly, PLX4720-induced hyperactivation of the MEK-ERK1/2 pathway promotes resistance to apoptosis in both non-invasive and invasive mutant N-RAS melanoma cells but does not enhance cell cycle properties. These findings underscore the need to genotypically stratify melanoma patients before enrollment on a mutant B-RAF inhibitor trial.

Topics: Apoptosis; Enzyme Activation; Genes, ras; Humans; Indoles; Melanoma; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides

This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN- cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Indoles; Melanoma; Membrane Proteins; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA, Small Interfering; Sulfonamides; Up-Regulation

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-Raf(V600E) mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf(V600E) also increases NHE1 activity that raises intracellular pH.

Topics: Amino Acid Substitution; Animals; Benzenesulfonates; Cation Transport Proteins; Cell Movement; Cell Size; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; HeLa Cells; Humans; Hydrogen-Ion Concentration; Indoles; Melanoma; Mutation, Missense; Niacinamide; Phenylurea Compounds; Phosphorylation; Protein Isoforms; Protein Kinase Inhibitors; Protein Structure, Tertiary; Proto-Oncogene Proteins B-raf; Pyridines; Rabbits; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sorafenib; Sulfonamides

Inhibitors of B-Raf and MEK kinases hold promise for the management of cutaneous melanomas harboring BRAF mutations. BRAF mutations are rare in uveal melanomas (UMs), but somatic mutations in the G protein α subunits Gαq and Gα11 (encoded by GNAQ and GNA11, respectively) occur in a mutually exclusive pattern in ∼80% of UMs. The impact of B-Raf and MEK inhibitors on Gα-mutant UMs remains unknown.. The impact of the B-Raf inhibitor PLX4720, the MEK inhibitor AZD6244, and the Akt inhibitor MK2206 on UM cell lines was assessed with the use of cell viability, proliferation, and apoptosis assays and immunoblot analysis.. BRAF-mutant UM cells were sensitive to both PLX4720 and AZD6244, undergoing cell cycle arrest but not apoptosis. UM cells with a Gα-protein mutation (GNAQ or GNA11) were mildly sensitive to AZD6244 but completely resistant to PLX4720. In fact, PLX4720 paradoxically increased ERK phosphorylation in Gα-mutant UM cells. The combination of AZD6244 with PLX4720 had synergistic anticancer activity in BRAF-mutant cells but not in Gα-mutant cells. The Akt inhibitor MK2206 sensitized BRAF-mutant cells to both PLX4720 and AZD6244 and sensitized Gα-mutant cells to AZD6244 but did not overcome the resistance of the Gα-mutant cells to PLX4720.. The response of UM cells to inhibition of B-Raf, MEK, and Akt depends on their genotype. Future use of such targeted therapies in clinical trials of UM patients will require careful design and patient selection based on genotype to provide personalized and effective therapy.

Topics: Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Cell Survival; Genotype; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; In Situ Nick-End Labeling; Indoles; MAP Kinase Kinase Kinases; Melanoma; Precision Medicine; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Sulfonamides; Uveal Neoplasms

Melanoma is a heterogeneous disease where monotherapies are likely to fail due to variations in genomic signatures. B-RAF inhibitors have been clinically inadequate but response might be augmented with combination therapies targeting multiple signaling pathways. We investigate the preclinical efficacy of combining the multikinase inhibitor sorafenib or the mutated B-RAF inhibitor PLX4720 with riluzole, an inhibitor of glutamate release that antagonizes metabotropic glutamate receptor 1 (GRM1) signaling in melanoma cells.. Melanoma cell lines that express GRM1 and either wild-type B-RAF or mutated B-RAF were treated with riluzole, sorafenib, PLX4720, or the combination of riluzole either with sorafenib or with PLX4720. Extracellular glutamate levels were determined by glutamate release assays. MTT assays and cell-cycle analysis show effects of the compounds on proliferation, viability, and cell-cycle profiles. Western immunoblotting and immunohistochemical staining showed apoptotic markers. Consequences on mitogen-activated protein kinase pathway were assessed by Western immunoblotting. Xenograft tumor models were used to determine the efficacy of the compounds in vivo.. The combination of riluzole with sorafenib exhibited enhanced antitumor activities in GRM1-expressing melanoma cells harboring either wild-type or mutated B-RAF. The combination of riluzole with PLX4720 showed lessened efficacy compared with the combination of riluzole and sorafenib in suppressing the growth of GRM1-expressing cells harboring the B-RAF(V600E) mutation.. The combination of riluzole with sorafenib seems potent in suppressing tumor proliferation in vitro and in vivo in GRM1-expressing melanoma cells regardless of B-RAF genotype and may be a viable therapeutic clinical combination.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Glutamic Acid; Indoles; Melanoma; Mice; Mice, Nude; Molecular Targeted Therapy; Mutation; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins B-raf; Pyridines; Receptors, Metabotropic Glutamate; Riluzole; Signal Transduction; Sorafenib; Sulfonamides; Xenograft Model Antitumor Assays

The initial use of BRAF targeted therapeutics in clinical trials has demonstrated encouraging responses in melanoma patients, although a rise in drug-resistant cells capable of advancing malignant disease has been described. The current study uses BRAFV600E expressing WM793 melanoma cells to derive data aimed at investigating the molecular determinant of cell invasion following treatment with clinical BRAF inhibitors.. Small-molecule inhibitors targeting BRAF reduced MEK1/2-ERK1/2 pathway activation and cell survival; yet, viable cell subpopulations persisted. The residual cells exhibited an elongated cell shape, prominent actin stress fibers and retained the ability to invade 3-D dermal-like microenvironments. BRAF inhibitor treatments were associated with reduced expression of RND3, an antagonist of RHOA activation, and elevated RHOA-dependent signaling. Restoration of RND3 expression or RHOA knockdown attenuated the migratory ability of residual cells without affecting overall cell survival. The invasive ability of BRAF inhibitor treated cells embedded in collagen gels was diminished following RND3 re-expression or RHOA depletion. Conversely, melanoma cell movement in the absence of BRAF inhibition was unaffected by RND3 expression or RHOA depletion.. These data reveal a novel switch in the requirement for RND3 and RHOA in coordinating the movement of residual WM793 cells that are initially refractive to BRAF inhibitor therapy. These results have important clinical implications because they suggest that combining BRAF inhibitors with therapies that target the invasion of drug-resistant cells could aid in controlling disease relapse.

Topics: Amino Acid Substitution; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Humans; Imidazoles; Indoles; Melanoma; Mutant Proteins; Neoplasm Invasiveness; Proto-Oncogene Proteins B-raf; Recombinant Fusion Proteins; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; RNA Interference; Signal Transduction; Spheroids, Cellular; Sulfonamides

Resistance to BRAF inhibitors is an emerging problem in the melanoma field. Strategies to prevent and overcome resistance are urgently required.. The dynamics of cell signalling, BrdU incorporation and cell-cycle entry after BRAF inhibition was measured using flow cytometry and western blot. The ability of combined BRAF/MEK inhibition to prevent the emergence of resistance was demonstrated by apoptosis and colony formation assays and in 3D organotypic cell culture.. BRAF inhibition led to a rapid recovery of phospho-ERK (pERK) signalling. Although most of the cells remained growth arrested in the presence of drug, a minor population of cells retained their proliferative potential and escaped from BRAF inhibitor therapy. A function for the rebound pERK signalling in therapy escape was demonstrated by the ability of combined BRAF/MEK inhibition to enhance the levels of apoptosis and abrogate the onset of resistance.. Combined BRAF/MEK inhibition may be one strategy to prevent the emergence of drug resistance in BRAF-V600E-mutated melanomas.

Topics: Apoptosis; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinases; Mutation; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Time Factors

Melanoma cells are highly resistant to anoikis, a form of apoptosis induced in nonadherent/inappropriate adhesion conditions. Depleting B-RAF or the prosurvival Bcl-2 family protein Mcl-1 renders mutant B-RAF melanoma cells susceptible to anoikis. In this study, we examined the effect of targeting B-RAF on the survival of primary stage melanoma cells cultured in three-dimensional type I collagen gels, which partially mimics the dermal microenvironment. Depletion/inhibition of B-RAF with small interfering RNA or the mutant B-RAF inhibitor, PLX4720, induced apoptosis of mutant B-RAF melanoma cells in three-dimensional collagen. Apoptosis was dependent on two upregulated BH3-only proteins, Bim-EL and Bmf, and was inhibited by ectopic Mcl-1 expression. Akt3 activation has been associated with the survival of melanoma cells. Mutant B-RAF melanoma cells ectopically expressing a constitutively activated form of Akt3 or endogenously expressing mutant Akt3 were protected from apoptosis induced by B-RAF knockdown or PLX4720 treatment. Furthermore, intrinsically resistant metastatic melanoma cells displayed elevated Akt phosphorylation in three-dimensional collagen and were rendered susceptible to PLX4720 by Akt3 knockdown. Importantly, myristylated Akt3 prevented B-RAF targeting-induced upregulation of Bim-EL and Bmf in three-dimensional collagen and partially protected Mcl-1-depleted cells from apoptosis. These findings delineate how mutant B-RAF protects melanoma cells from apoptosis and provide insight into possible resistance mechanisms to B-RAF inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line, Tumor; Humans; Indoles; Melanoma; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Up-Regulation

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, Bim(S), remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF(V600E) triggers preferential splicing to produce Bim(S), which is particularly important in induction of apoptosis in B-RAF(V600E) melanoma cells. Although the specific B-RAF(V600E) inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim(EL), Bim(L), and Bim(S), at the protein and mRNA levels in B-RAF(V600E) melanoma cells, the increase in the ratios of Bim(S) mRNA to Bim(EL) and Bim(L) mRNA indicates that it favours Bim(S) splicing. Consistently, enforced expression of B-RAF(V600E) in wild-type B-RAF melanoma cells and melanocytes inhibits Bim(S) expression. The splicing factor SRp55 appears necessary for the increase in Bim(S) splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim(S) and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim(S) splicing is also mirrored in freshly isolated B-RAF(V600E) melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF(V600E) inhibitors.

Topics: Amino Acid Substitution; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line, Tumor; Humans; Indoles; Melanoma; Membrane Proteins; Mutation; Nuclear Proteins; Phosphoproteins; Protein Isoforms; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; RNA Interference; RNA Splicing; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Serine-Arginine Splicing Factors; Sulfonamides

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Escherichia coli; Humans; Indoles; Inhibitory Concentration 50; Melanoma; Mice; Mice, SCID; Models, Molecular; Molecular Structure; Oncogenes; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides

Topics: Antineoplastic Agents; Benzenesulfonates; Clinical Trials as Topic; Drug Delivery Systems; Drug Evaluation, Preclinical; Humans; Indoles; Melanoma; Niacinamide; Phenylurea Compounds; Point Mutation; Proto-Oncogene Proteins B-raf; Pyridines; Sorafenib; Sulfonamides; Treatment Outcome