Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines

Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.

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Rho-mediated gene transcription promotes BRAF inhibitor resistance in de-differentiated melanoma cells

10.1101/381806 ◽

2018 ◽

Cited By ~ 1

Author(s):

SA Misek ◽

KM Appleton ◽

TS Dexheimer ◽

EM Lisabeth ◽

RS Lo ◽

...

Keyword(s):

Cell Lines ◽

Gene Transcription ◽

Melanoma Cell ◽

Cutaneous Melanoma ◽

Acquired Resistance ◽

Rho Kinase ◽

Braf Inhibitor ◽

Resistant Cell ◽

Melanoma Cell Lines

AbstractOver half of cutaneous melanoma tumors have BRAFV600E/Kmutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that approximately 50-60% of melanoma cell lines with vemurafenib resistance acquiredin vitroshow activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anti-cancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHighBRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapy approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

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Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

BMC Cancer ◽

10.1186/s12885-021-08607-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Kana Oiwa ◽

Naoko Hosono ◽

Rie Nishi ◽

Luigi Scotto ◽

Owen A. O’Connor ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Acquired Resistance ◽

3D Culture ◽

Resistance Mechanisms ◽

Resistant Cell ◽

Gene Set Enrichment Analysis ◽

Synergistic Activity ◽

Mechanisms Of Resistance ◽

Resistant Cells

Abstract Background Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. Methods To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. Results Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. Conclusions The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

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Efficacy of the CDK4/6 Dual Inhibitor Abemaciclib in EGFR-Mutated NSCLC Cell Lines with Different Resistance Mechanisms to Osimertinib

Cancers ◽

10.3390/cancers13010006 ◽

2020 ◽

Vol 13 (1) ◽

pp. 6

Author(s):

Silvia La Monica ◽

Claudia Fumarola ◽

Daniele Cretella ◽

Mara Bonelli ◽

Roberta Minari ◽

...

Keyword(s):

Cell Lines ◽

Kinase Inhibitor ◽

Acquired Resistance ◽

Growth Factor Receptor ◽

Resistance Mechanisms ◽

Resistant Cell ◽

Nsclc Cell ◽

Dual Inhibitor ◽

Nsclc Patients ◽

Egfr Mutated

Abemaciclib is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6 that inhibits the transition from the G1 to the S phase of the cell cycle by blocking downstream CDK4/6-mediated phosphorylation of Rb. The effects of abemaciclib alone or combined with the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib were examined in a panel of PC9 and HCC827 osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines carrying EGFR-dependent or -independent mechanisms of intrinsic or acquired resistance. Differently from sensitive cells, all the resistant cell lines analyzed maintained p-Rb, which may be considered as a biomarker of osimertinib resistance and a potential target for therapeutic intervention. In these models, abemaciclib inhibited cell growth, spheroid formation, colony formation, and induced senescence, and its efficacy was not enhanced in the presence of osimertinib. Interestingly, in osimertinib sensitive PC9, PC9T790M, and H1975 cells the combination of abemaciclib with osimertinib significantly inhibited the onset of resistance in long-term experiments. Our findings provide a preclinical support for using abemaciclib to treat resistance in EGFR mutated NSCLC patients progressed to osimertinib either as single treatment or combined with osimertinib, and suggest the combination of osimertinib with abemaciclib as a potential approach to prevent or delay osimertinib resistance in first-line treatment.

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Abstract 2399: System analysis of adapted and quiescent/dormancy(QD) models of drug resistance in melanoma cell lines

10.1158/1538-7445.am2016-2399 ◽

2016 ◽

Author(s):

Feng Liu ◽

Parvita Paresh Panchal ◽

Zi Wang ◽

Ahmed Farhat ◽

Angela Garcia ◽

...

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Melanoma Cell ◽

System Analysis ◽

Melanoma Cell Lines

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Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

Cells ◽

10.3390/cells9010142 ◽

2020 ◽

Vol 9 (1) ◽

pp. 142 ◽

Cited By ~ 11

Author(s):

Mariusz L. Hartman ◽

Malgorzata Sztiller-Sikorska ◽

Anna Gajos-Michniewicz ◽

Malgorzata Czyz

Keyword(s):

Phenotypic Plasticity ◽

Cell Lines ◽

Mapk Pathway ◽

Acquired Resistance ◽

Resistance Mechanisms ◽

Genetic Alterations ◽

Resistant Cell ◽

Cross Resistance ◽

Preclinical Model ◽

Development Of Resistance

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

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nmd , a novel gene differentially expressed in human melanoma cell lines, encodes a new atypical member of the enzyme family of lipases

FEBS Letters ◽

10.1016/s0014-5793(97)00098-7 ◽

1997 ◽

Vol 404 (1) ◽

pp. 82-86 ◽

Cited By ~ 14

Author(s):

Jan J.M. van Groningen ◽

Maarten R. Egmond ◽

Henri P.J. Bloemers ◽

Guido W.M. Swart

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Human Melanoma ◽

Differentially Expressed ◽

Human Melanoma Cell ◽

Novel Gene ◽

Enzyme Family ◽

Atypical Member ◽

Melanoma Cell Lines

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Vemurafenib resistant melanoma cells acquire mesenchymal stem cell-like properties

Advances in molecular oncology ◽

10.17650/2313-805x-2019-6-4-47-57 ◽

2019 ◽

Vol 6 (4) ◽

pp. 47-57

Author(s):

A. A. Vartanian ◽

O. S. Burova ◽

Kh. S. Vishnyakova ◽

I. V. Samoylenko ◽

V. A. Misyurin ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Acquired Resistance ◽

Melanoma Cells ◽

Mapk Signaling ◽

Brafv600e Mutation ◽

Acquired Drug Resistance ◽

Melanoma Patients ◽

Braf Mutant ◽

Melanoma Cell Lines

Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months. Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance.Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study.Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells. Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

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Role of AXL in metastatic melanoma and impact of TP-0903 as a novel therapeutic option for melanoma brain metastasis.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e22021 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e22021-e22021

Author(s):

Eemon Tizpa ◽

Hannah J Young ◽

Kimberley-Jane C. Bonjoc ◽

Chou-Wei Chang ◽

Yilun Liu ◽

...

Keyword(s):

Overall Survival ◽

Drug Resistance ◽

Malignant Melanoma ◽

Cell Viability ◽

Cell Lines ◽

Melanoma Cell ◽

Therapeutic Option ◽

Poor Survival ◽

Malignant Melanoma Cell ◽

Melanoma Cell Lines

e22021 Background: Melanoma brain metastases (MBM) are common with a median overall survival of 4-5 months. Although immunotherapies have improved clinical outcomes and have doubled overall survival in MBM, there is a high incidence rate of relapse caused by drug resistance. AXL, a receptor tyrosine kinase (RTK), is associated with drug resistance and metastasis in many cancers. The activation of AXL via trans-phosphorylation regulates multiple signaling pathways that induce tumor survival, metastasis, drug resistance, and epithelial-to-mesenchymal transition (EMT). In MBM, AXL is upregulated and associated with disease progression, promoting cell invasion and migration. This suggests that targeting AXL can be a novel strategy to overcome treatment-related resistance in MBM. TP-0903, an investigational small molecule inhibitor of AXL, has shown efficacy in reversing the mesenchymal phenotype and re-sensitizing resistant cancer cells to targeted therapies in heme malignancies, pancreatic, and breast cancer. We aim to investigate the efficacy of TP-0903 in MBM. Methods: The Cancer Genome Atlas (TCGA) data was utilized to investigate the signaling pathways downstream of AXL that are upregulated in advanced melanoma. Nine signaling molecules including AKT1, mTOR, and PAK4 were analyzed to identify any correlation between gene expression levels and overall survival. Four metastatic melanoma cell lines were used to evaluate the effect of TP-0903 on cell viability and active AXL downregulation was assessed in vitro through MTS cell viability assays and Immunoblotting. Wound closure assays were executed to understand the functional consequences of AXL downregulation. Results: In all nine genes, high expression levels confer poor survival probability. Cell viability assays of four malignant melanoma cell lines showed that TP-0903 treatment resulted in IC50 values ranging from 32 – 692 nM. Western blot analysis indicated that TP-0903 reduced the levels of phosphorylated AXL in malignant melanoma cell lines. In addition, increasing TP-0903 concentrations reduced the rate of cell migration in these malignant melanoma cell lines. Conclusions: AXL plays a role in EMT, treatment resistance, and metastasis in MBM, resulting in poor survival. Our findings suggest TP-0903 is effective in reducing cell migration, inhibit metastasis, and can be a potential therapeutic option in MBM.

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