plx-4720 and Skin-Neoplasms

Therapeutic success of targeted therapy with BRAF inhibitors (BRAFi) for melanoma is limited by resistance development. Observations from preclinical mouse models and recent insights into the immunological effects caused by BRAFi give promise for future development of combination therapy for human melanoma. In our study, we used the transplantable D4M melanoma mouse model with the BRAF

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Imiquimod; Indoles; Killer Cells, Natural; Lymphocytes, Tumor-Infiltrating; Male; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mutation; Natural Killer T-Cells; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Sulfonamides; T-Lymphocytes; Toll-Like Receptor 7

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

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

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

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

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

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

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

B-Raf(V600E) inhibitors have been suggested to promote tumor regression with the help of host immunity, but this hypothesis has not been examined directly in detail. In this study, we profiled immunologic changes in the tumor microenvironment and tumor-infiltrating lymphocytes (TIL) in a B-RafV600E/Pten-driven murine model of melanoma after administration of the B-Raf(V600E) small molecule inhibitor PLX4720. In this model, we found that as tumors developed, they gradually acquired immunosuppressive features, including accumulation of regulatory T cells (Treg) and CD11b(+)/Gr-1(+) myeloid cells and loss of Th1 effector functions on CD4(+) TILs, such as CD40L and IFNγ expression. PLX4720 administration promoted development of a more immune stimulatory microenvironment associated with a relative increase in CD40L and IFNγ expression on intratumoral CD4(+) TILs and a reduced accumulation of Tregs and CD11b(+)/Gr-1(+) myeloid cells. Strikingly, CD40L or IFNγ blockade compromised the ability of PLX4720 to inhibit melanoma growth. Supporting this result, agonistic CD40 antibody was sufficient to evoke antitumor immunity and suppress tumor growth in tumor-bearing mice. Taken together, our results establish the critical role of immune-related changes, with key contributions for CD40L and IFNγ signaling in the antitumor responses triggered in vivo by B-Raf(V600E) inhibitors.

Topics: Animals; Antigen-Presenting Cells; Antineoplastic Agents; CD4-Positive T-Lymphocytes; CD40 Ligand; Drug Screening Assays, Antitumor; Indoles; Interferon-gamma; Macrophages; Melanoma, Experimental; Mice; Mice, Transgenic; Mutation, Missense; Proto-Oncogene Proteins B-raf; Signal Transduction; Skin Neoplasms; Sulfonamides; Tumor Microenvironment

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

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

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

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

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