scholarly journals Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance

2021 ◽  
Vol 17 (11) ◽  
pp. e1009515
Author(s):  
Mathurin Dorel ◽  
Bertram Klinger ◽  
Tommaso Mari ◽  
Joern Toedling ◽  
Eric Blanc ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Proteomic Profiling ◽  
Mek Inhibitors ◽  
Mapk Signalling ◽  
Perturbation Data ◽  
Specific Feedback ◽  
Signalling Models

Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma.

scholarly journals Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance

2021 ◽  
Author(s):  
Mathurin Dorel ◽  
Bertram Klinger ◽  
Tommaso Mari ◽  
Joern Toedling ◽  
Eric Blanc ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Mek Inhibitors ◽  
Mapk Signalling ◽  
Perturbation Data ◽  
Specific Feedback ◽  
Quantitative Understanding ◽  
Signalling Models

Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and to the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospo-proteomics profiles confirm the cell-specific feedback effects and synergy of MEK and IGFR targeted treatements. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies, and our findings should be considered for the planning of future clinical trials introducing MEK inhibitors in the treatment of neuroblastoma.

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

Cancers ◽  
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.

scholarly journals 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 ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 142 ◽  
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.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

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

BMC Cancer ◽  
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.

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

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6058
Author(s):  
Vikas Patel ◽  
István Szász ◽  
Viktória Koroknai ◽  
Tímea Kiss ◽  
Margit Balázs
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Differentially Expressed ◽  
Molecular Alterations ◽  
Melanoma Cell Lines ◽  
Resistant Cells

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.

scholarly journals IGF1R/IR Mediates Resistance to BRAF and MEK Inhibitors in BRAF-Mutant Melanoma

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5863
Author(s):  
Hima Patel ◽  
Rosalin Mishra ◽  
Nour Yacoub ◽  
Samar Alanazi ◽  
Mary Kate Kilroy ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Treatment Options ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Sequencing Analysis ◽  
Mek Inhibitors ◽  
Braf Mutant

The use of BRAF and MEK inhibitors for patients with BRAF-mutant melanoma is limited as patients relapse on treatment as quickly as 6 months due to acquired resistance. We generated trametinib and dabrafenib resistant melanoma (TDR) cell lines to the MEK and BRAF inhibitors, respectively. TDR cells exhibited increased viability and maintenance of downstream p-ERK and p-Akt as compared to parental cells. Receptor tyrosine kinase arrays revealed an increase in p-IGF1R and p-IR in the drug resistant cells versus drug sensitive cells. RNA-sequencing analysis identified IGF1R and INSR upregulated in resistant cell lines compared to parental cells. Analysis of TCGA PanCancer Atlas (skin cutaneous melanoma) showed that patients with a BRAF mutation and high levels of IGF1R and INSR had a worse overall survival. BMS-754807, an IGF1R/IR inhibitor, suppressed cell proliferation along with inhibition of intracellular p-Akt in TDR cells. Dual inhibition of IGF1R and INSR using siRNA reduced cell proliferation. The combination of dabrafenib, trametinib, and BMS-754807 treatment reduced in vivo xenograft tumor growth. Examining the role of IGF1R and IR in mediating resistance to BRAF and MEK inhibitors will expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients.

scholarly journals Drivers of AR indifferent anti-androgen resistance in prostate cancer cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Florian Handle ◽  
Stefan Prekovic ◽  
Christine Helsen ◽  
Thomas Van den Broeck ◽  
Elien Smeets ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
Parp Inhibitor ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Castration Resistant Prostate Cancer ◽  
Tissue Samples ◽  
Cellular Models ◽  
Castration Resistant

Abstract Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.

scholarly journals A Study of Multiple Drug Resistance Mechanisms Improved Against Bortezomib on Multiple Myeloma Cell Lines In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4183-4183
Author(s):  
Tolga Uyuklu ◽  
A. Ugur Ural ◽  
Meral Sarper ◽  
Ferit Avcu ◽  
Yusuf Baran ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Multi Drug Resistance ◽  
Resistance Protein ◽  
One Year ◽  
Rpmi 8226

Abstract The most important problem in the treatment of Multiple Myeloma (MM) is the multi drug resistance (MDR) observed before and after the treatment. For this reason in MM cases an early resistance to treatment can be developed or the disease can relapsed in early period. Yet, there has been no improved drug resistance against proteazom inhibitor Bortezomib (Bor), which is used alone or with other chemotherapeutic agents in resistant or relapsed MM cases. In this study, bortezomib resistant human MM cell lines; RPMI-8226, secreting lambda light chain, and ARH-77, secreting IgG, were developed and responsible resistance mechanisms were investigated. For this purpose, by exposing to the cells to sequentially gradual doses of Bor in vitro conditions, resistant cell lines were acquired throughout one year. The IC50 values for Bor were determined after 48 hour incubation by MTT cytotoxicity assay (IC50:1,16nM for RPMI-8226 and IC80:0,6nM for ARH-77) against wild type cells. Throughout one year some cell lines resistant to 1,3nM Bor were acquired by performing Bor to both cell lines in gradual doses. In resistant cell lines IC50:18,07 for RPMI-8226 and IC50:97,56 nM for ARH-77 were determined by MTT assay. In parallel of the gradual increase in drug concentration; the expression changes of the genes of ATP binding cassette protein; MDR1 (Multi Drug Resistance Protein), MRP1 (Multi Drug Resistance Associated Protein), BCRP (Breast Cancer Resistance Protein); and LRP (Lung Resistant Protein) which is responsible for accumulation of the drug in cytoplasm with the aid of nuclear membrane were determined with Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and densitometric analysis. In resistant cells, high expression of MDR1, MRP1, BRCP and LRP genes showed that; pumping the drug out of the cell membrane and decrease in accumulation of the drug in the cytoplasm had effects on the resistant mechanisms against Bor. Furthermore, expression changes of an important sing of apoptosis ‘caspase-3’, pro-apoptotic ‘bax’ and an anti-apoptotic ‘bcl-2’ genes were examined by RT-PCR and we could come to a point that when compared the sensitive cells to resistant cells, expression of caspase-3 gene and pro-apoptotic bax protein decreased but bcl-2 gene expression increased in resistant cell lines. Finally, we concluded that resistant cell lines acquired resistance against apoptosis by means of mitochondria. By means of this project, the genes which are responsible for secondary drug resistance in ARH-77 and RPMI-8226 MM cell lines in vitro conditions against Bor were determined. Also resistance mechanisms against apoptosis were demonstrated. Cross resistance to different chemotherapeutic agents mechanisms are still continuing.

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