Project description:Whole-exome sequencing was performed on DNA samples extracted from seven melanoma cell lines resistant to either vemurafenib (BRAF V600E inhibitor) or trametinib (MEK1/2 inhibitor). The aim of the experiment was to search for genetic alterations responsible for phenotypic diversity of melanoma cell lines reported at the level of cell morphology, activity of signaling pathways essential for melanoma development and progression, and resistance to targeted therapeutics.
Project description:Whole-exome sequencing was performed on DNA samples extracted from seven melanoma cell lines resistant to either vemurafenib (BRAF V600E inhibitor) or trametinib (MEK1/2 inhibitor). The aim of the experiment was to search for genetic alterations responsible for phenotypic diversity of melanoma cell lines reported at the level of cell morphology, activity of signaling pathways essential for melanoma development and progression, and resistance to targeted therapeutics.
Project description:Whole-exome sequencing of genomic DNA extracted from patient-derived melanoma cell lines resistant to vemurafenib (PLX) or trametinib (TRA)
Project description:Whole-exome sequencing was performed on DNA sample extracted from one melanoma cell line resistant to vemurafenib (BRAF V600E inhibitor). The aim of the experiment was to search for genetic alterations responsible for phenotypic diversity of melanoma cell lines reported at the level of cell morphology, activity of signaling pathways essential for melanoma development and progression, and resistance to targeted therapeutics.
Project description:The majority of human melanomas bears BRAF mutations and thus is treated with inhibitors of BRAF, such as vemurafenib. While patients with BRAF mutations often demonstrate an initial dramatic response to vemurafenib, relapse is extremely common. Thus, novel agents are needed for the treatment of these aggressive melanomas. Honokiol is a small molecule compound derived from Magnolia grandiflora that has activity against solid tumors and hematopoietic neoplasms. In order to increase the lipophilicity of honokiol, we have synthesized honokiol DCA, the dichloroacetate ester of honokiol. In addition, we synthesized a novel fluorinated honokiol analog, bis-trifluoromethyl-bis-(4-hydroxy-3-allylphenyl) methane (hexafluoro). Both compounds exhibited activity against A375 melanoma in vivo, but honokiol DCA was more active. Gene arrays comparing treated with vehicle control tumors demonstrated induction of the respiratory enzyme succinate dehydrogenase B (SDHB) by treatment, suggesting that our honokiol analogs induce respiration in vivo. We then examined its effect against a pair of melanomas, LM36 and LM36R, in which LM36R differs from LM36 in that LM36R has acquired vemurafenib resistance. Honokiol DCA demonstrated in vivo activity against LM36R (vemurafenib resistant) but not against parental LM36. Honokiol DCA and hexafluoro inhibited the phosphorylation of DRP1, thus stimulating a phenotype suggestive of respiration through mitochondrial normalization. Honokiol DCA may act in vemurafenib resistant melanomas to increase both respiration and reactive oxygen generation, leading to activity against aggressive melanoma in vivo.
Project description:The majority of human melanomas bears BRAF mutations and thus is treated with inhibitors of BRAF, such as vemurafenib. While patients with BRAF mutations often demonstrate an initial dramatic response to vemurafenib, relapse is extremely common. Thus, novel agents are needed for the treatment of these aggressive melanomas. Honokiol is a small molecule compound derived from Magnolia grandiflora that has activity against solid tumors and hematopoietic neoplasms. In order to increase the lipophilicity of honokiol, we have synthesized honokiol DCA, the dichloroacetate ester of honokiol. In addition, we synthesized a novel fluorinated honokiol analog, bis-trifluoromethyl-bis-(4-hydroxy-3-allylphenyl) methane (hexafluoro). Both compounds exhibited activity against A375 melanoma in vivo, but honokiol DCA was more active. Gene arrays comparing treated with vehicle control tumors demonstrated induction of the respiratory enzyme succinate dehydrogenase B (SDHB) by treatment, suggesting that our honokiol analogs induce respiration in vivo. We then examined its effect against a pair of melanomas, LM36 and LM36R, in which LM36R differs from LM36 in that LM36R has acquired vemurafenib resistance. Honokiol DCA demonstrated in vivo activity against LM36R (vemurafenib resistant) but not against parental LM36. Honokiol DCA and hexafluoro inhibited the phosphorylation of DRP1, thus stimulating a phenotype suggestive of respiration through mitochondrial normalization. Honokiol DCA may act in vemurafenib resistant melanomas to increase both respiration and reactive oxygen generation, leading to activity against aggressive melanoma in vivo. Tumors from animals treated with control vehicle, honokiol DCA, and hexafluoro were harvested and snapped-frozen in liquid nitrogen until RNA Extraction. RNA extraction was performed according to the Qiagen RNeasy kit. RNA samples were then submitted to Emory Universityâs Intergrated Genomics Core for RNA quality analysis and gene expression assay. Gene expression analysis was performed using an Illumina HumanHT-12 v3 Expression Bead Chip and Gene Expression Module of Illuminaâs GenomeStudio Software package (v2011.1, Illumina).
Project description:RNA-seq data were generated for two conditions: for parental SK-MEL-239 cells grown in normal media and resistant SK-MEL-239 cells grown in media supplemented with vemurafenib.
Project description:The development of targeted therapies (Braf/MEK inhibitors) and immunotherapy have had a major impact on the treatment of melanoma. However, the majority of patients with advanced melanomas succumb to their disease. The mechanisms of resistance to both targeted therapies and immunotherapies are numerous and have been well-described. These include the alternative acti-vation of Braf/MEK signaling, novel compensating mutations in additional oncogenes, and loss of neoantigens. There has been limited development of small molecules that target alternative path-ways in melanoma in the last two decades. We have previously identified triphenylmethanes as a class that shows activity against a wide variety of tumors. We have synthesized a novel triphenyl-methane, indolium 1, and demonstrated its efficacy against an aggressive vemurafenib-resistant melanoma in vivo. Indolium 1 has a novel mechanism of action against melanoma, in that it results in induction of the tumor-suppressor EphA3. We believe that pre-IND studies are warranted for this novel compound, given its mechanism of action and ability to inhibit the growth of vemurafenib resistant melanoma in vivo
Project description:BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current front-line therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus, and a missense point mutation of the transcriptional repressor BCORL1, in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a change-of-function mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.
Project description:To explore the characteristics of senescent melanoma cells induced by vemurafenib or cisplatin, melanoma A375 cells were treated with vemurafenib and cisplatin, respectively. The senescent phenotypes were verified by β-gal staining, EdU assay, cell morphology and the senescence-related pathways. RNA-seq was performed to explore the differentially expressed genes in the senescent cells induced by vemurafenib or cisplatin.