Drug-resistant Melanoma Cells Research Articles

Second-Generation Cap Analogue Prodrugs for Targeting Aberrant Eukaryotic Translation Initiation Factor 4E (eIF4E) Activity in Drug-Resistant Melanoma.

Melanoma is the deadliest form of skin cancer with a 5-year survival rate of less than 20%. While significant strides have been made in the field of kinase-targeted and immune-based therapies for melanoma, the development of resistance to these therapeutic agents has hindered the success of treatment. Drug-resistant melanoma is particularly reliant on enhanced cap-dependent translation to drive the production of oncoproteins that promote growth and survival. The m7GpppX cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor of cap-dependent translation initiation, and its overexpression in melanoma tumors has been shown to drive resistance to BRAFV600E kinase-targeted inhibitors. These findings point to eIF4E-targeted therapies as a promising strategy to overcome drug resistance in melanoma. Herein, we build upon our previous work of developing cell-permeable cap analogue inhibitors to design second-generation cap analogues that inhibit eIF4E-mediated cap-dependent translation in drug-resistant melanoma cells.

Cyclic tachyplesin I kills proliferative, non-proliferative and drug-resistant melanoma cells without inducing resistance

Acquired drug resistance is the major cause for disease recurrence in cancer patients, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. To address this problem, we investigated cyclic membrane-active peptides as an alternative therapeutic modality to kill drug-tolerant and resistant melanoma cells to avoid acquired drug resistance. We selected two stable cyclic peptides (cTI and cGm), previously shown to have anti-melanoma properties, and compared them with dabrafenib, a drug used to treat cancer patients with the BRAF V600E mutation. The peptides act via a fast membrane-permeabilizing mechanism and kill metastatic melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Melanoma cells do not become resistant to long-term treatment with cTI, nor do they evolve their lipid membrane composition, as measured by lipidomic and proteomic studies. In vivo studies in mice demonstrated that the combination treatment of cTI and dabrafenib resulted in fewer metastases and improved overall survival. Such cyclic membrane-active peptides are thus well suited as templates to design new anticancer therapeutic strategies.

FOXN3 Regulates Autophagic Activity to Suppress Drug Resistance in Melanoma Cells.

The forkhead box (FOX) family member FOXN3has been reported to inhibit transcriptional activity associated with regulating tumor development. However, the role of FOXN3 in the pathogenesis of melanoma is not well understood. To investigate the biological functions of FOXN3 in drug resistance of melanoma. The expression of FOXN3 in melanoma was investigated using Gene Expression profiling interactive analysis (GEPIA) and Linkedomics databases. Melanoma cell proliferation, invasion, and migration were assessed using the colony formation assay, the scratch wound healing test, the Transwell invasion assay, and the nude mice xenograft to determine the effects of FOXN3 over-expression and depletion. The functional role of the transcriptional regulator in melanoma cells was tested through chromatin immunoprecipitation, immunofluorescence. FOXN3 was downregulated in melanoma. Over-expression of FOXN3 inhibited the proliferation and motility of melanoma cells, whereas FOXN3 knockdown significantly enhanced the proliferation and motility of melanoma cells. Overexpression of FOXN3 reduced autophagic activity in melanoma cells. Enhanced autophagic activity in drug-resistant melanoma cell lines is related to drug-sensitive cells, and significant differences in FOXN3 localization were observed when comparing melanoma cells that were sensitive and resistant to Vemurafenib. Additionally, FOXN3has been identified as binding to the promoter region of the cancer antigen Fibrous Sheath Interacting Protein 1 (FSIP1), thereby regulating the expression of this gene. FOXN3 functions as an important regulator of the development and progression of Vemurafenib-resistant melanoma cells, partly owing to its binding to the FISP1. As such, FOXN3 may represent a relevant target for therapeutic interventions in patients suffering from drug-resistant melanoma.

Melanoma redox biology and the emergence of drug resistance.

Melanoma is the deadliest form of skin cancer, with the loss of approximately 60,000 lives world-wide each year. Despite the development of targeted therapeutics, including compounds that have selectivity for mutant oncoproteins expressed only in cancer cells, many patients are either unresponsive to initial therapy or their tumors acquire resistance. This results in five-year survival rates of below 25%. New strategies that either kill drug-resistant melanoma cells or prevent their emergence would be extremely valuable. Melanoma, like other cancers, has long been described as being under increased oxidative stress, resulting in an increased reliance on antioxidant defense systems. Changes in redox homeostasis are most apparent during metastasis and during the metabolic reprogramming associated with the development of treatment resistance. This review discusses oxidative stress in melanoma, with a particular focus on targeting antioxidant pathways to limit the emergence of drugresistant cells.

Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin

BackgroundBRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488.MethodsInvasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses.ResultsWe demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients.ConclusionsAltogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions.Graphical

PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.

Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation. The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance. Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance. Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.

Abstract 411: PHI-501, a novel pan-RAF/DDRs dual kinase inhibitor, overcomes BRAF or MEK inhibitor resistance in melanoma

Abstract Background: PHI-501 is a newly developed, highly effective, and orally accessible dual inhibitor for pan-RAF and discoidin domain receptor (DDR) that is a collagen-activated receptor tyrosine kinase. Mutations of the BRAF and NRAS are the most common genetic alterations in melanoma and consequently lead to activation of mitogen activated protein kinase (MAPK) signaling. The major drawback of melanoma treatment is the innate and acquired drug resistance to MAPK inhibitors. In this study, we investigated the inhibitory effects of a novel compound, PHI-501 on resistance to known BRAF and MEK inhibitors in melanoma. Methods: After compounds treatment, the growth-inhibiting activity was assessed using the CCK test. Inhibition of RAF/DDR1 pathway signaling was evaluated by western blotting. Using annexin-V/propidium iodide-stained cells and flow cytometry, apoptosis induction was assessed. To quantify cell mobility, monolayer cells were subjected to a wound-healing assay. Long-term therapy with dabrafenib (BRAF inhibitor), cobimetinib or trametinib (MEK inhibitor) led to the establishment of the drug-resistant SK-MEL-3 (BRAF V600E) and SK-MEL-30 (NRAS Q61K) melanoma cell line. Results: PHI-501 demonstrated potent growth inhibition (GI50 <1 µM) in seven melanoma cell lines harboring BRAF V600E or NRAS mutations. Western blot revealed that PHI-501 inhibited the phosphorylation of MEK, ERK, and AKT proteins in the downstream signaling of RAF/DDRs and decreased the levels of cyclin D1 and survivin. In SK-MEL-3, PHI-501 was 47-fold more effective than the class I BRAF inhibitor, dabrafenib (GI50: 0.08 µM vs 3.73 µM). Established dabrafenib- or trametinib-resistant SK-MEL-3 cells were extremely sensitive to PHI-501 (GI50: 0.20 µM and 0.13 µM), whereas their sensitivity to dabrafenib or trametinib was diminished (GI50: NA and 39.61 µM). PHI-501 triggered apoptosis more effectively than these inhibitors, as revealed by a larger proportion of annexin staining and extensive PARP1 cleavage. Comparing the potency of PHI-501 to cobimetinib in acquired resistant SK-MEL-30 cells showed that PHI-501 had 74-fold more inhibitory effect on cell proliferation (GI50: 0.55 µM to 40 µM). Moreover, treatment with PHI-501 strongly suppressed cell migration of cobimetinib-resistant SK-MEL-30 cells. PHI-501 inhibited the growth of drug-resistant melanoma cells as effectively as the original drug-naïve cells, and western blot analysis revealed considerable suppression of DDR1/2, ERK, and AKT phosphorylation in MAPK inhibitor-resistant cells. Conclusion: Melanoma cells resistant to RAF or MEK-targeted treatments or harboring NRAS mutation exhibited strong antiproliferative activity when treated with PHI-501, a highly potent pan-RAF/DDR dual inhibitor. The results of this study suggest that PHI-501, as a single agent, has the potential to overcome the restricted response in the treatment of melanoma. Citation Format: Sue Min Kim, Jung Hee Park, Ky-Youb Nam, June H-J Han, Kyu-Tae Kim, Jeong Hyeok Yoon, Sandip Sengupta, Taebo Sim, Sang Joon Shin. PHI-501, a novel pan-RAF/DDRs dual kinase inhibitor, overcomes BRAF or MEK inhibitor resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 411.

Combining fiber optical tweezers and Raman spectroscopy for rapid identification of melanoma.

Cutaneous melanoma is a skin tumor with a high degree of malignancy and fatality rate, the incidence of which has increased in recent years. Therefore, a rapid and sensitive diagnostic technique of melanoma cells is urgently needed. In this paper, we present a new approach using fiber optical tweezers to manipulate melanoma cells to measure their Raman spectra. Then, combined with Principal Component Analysis and Support Vector Machines (PCA-SVM) classification model, to achieve the classification of common mutant, wild-type and drug-resistant melanoma cells. A total of 150 Raman spectra of 30 cells were collected from mutant, wild-type and drug-resistant melanoma cell lines, and the classification accuracy was 92%, 94%, 97.5%, respectively. These results suggest that the study of tumor cells based on fiber optical tweezers and Raman spectroscopy is a promising method for early and rapid identification and diagnosis of tumor cells.

Abstract 348: Efficacy of triple combination treatment with trametinib, mebendazole and CD133 RNA aptamer in recalcitrant NRAS-mutant melanoma cells

Abstract Malignant melanoma is the most aggressive form of skin cancer. Although kinase inhibitors have proven to be efficient with promising clinical results, the development of drug resistance is one of the major issues in targeted therapy. CD133, also known asProminin-1, is a membrane glycoprotein studied extensively as a cell surface marker for cancer stem cells. In our previous studies, mebendazole, a repurposed broad spectrum anti-helminthic agent, inhibited the growth of NRAS mutant melanoma cells in combination with trametinib, a MEK inhibitor. However, a CD133-positive cancer stem cell population remain resistant to this targeted therapy. Use of RNA aptamers in diagnostic as well as therapeutics has been studied recently in different types of cancer cells. Drug delivery of aCD133 aptamer coupled with doxorubicin has been found to inhibit the growth of liver cancer cells. In this study, we targeted the cancer stem cells expressing CD133 with RNA aptamers, in combination with trametinib and mebendazole to sensitize the cells to the combination treatment. Patient derived melanoma cell lines, BAKP and POT, harboring BRAFWT/NRASQ61K and BRAFWT/NRASQ61R driver mutations, were co-transduced with aTet-on vector expressing CD133 and a Tet activator. The doxycycline-induced and uninduced cells were subjected to single agent treatment with 100 nM trametinib, 100 nM mebendazole or 100 nM CD133 RNA aptamer, as well as dual and triple agent combinations for 24 or 48 hours. XTT cell viability and apoptotic assays such as AnnexinV-PI flow cytometry analysis revealed that the triple combination with trametinib, mebendazole and the CD133 aptamer was the most effective in inducing apoptotic cell death, even in the doxycycline-induced CD133-overexpressing melanoma cells. Further studies will use preclinical in vivo mouse xenograft models to investigate the effects on tumor growth of CD133 RNA aptamers in combination treatments, potentially making it available for clinical trials aiming to improve patient response to therapeutics against drug-resistant melanoma cells. Citation Format: Yoga Haribabu, Raymond Chien, Ryyan Alobaidi, Li-Wei Kuo, Nusrat Islam, Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal. Efficacy of triple combination treatment with trametinib, mebendazole and CD133 RNA aptamer in recalcitrant NRAS-mutant melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 348.

UPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells.

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients’ outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenib-resistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.

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

Simple SummaryMelanoma is the most aggressive, deadliest form of skin cancer. Combined BRAF-MEK inhibitor (BRAFi/MEKi) therapy was a breakthrough in the treatment of melanoma with BRAFV600-mutations. However, many patients frequently develop drug resistance to the combinatory treatment. The aim of our study was to characterize the molecular background behind acquired resistance to BRAFi/MEKi-s. After the successful development of resistant cell lines, we investigated the invasion properties, changes in gene and protein expressions, as well as the effect of the “drug holiday” of the resistant cell lines. Drug-resistant melanoma cells had a higher invasive potential and acquired a spindle-like structure, and many cancer-related proteins were overexpressed in the resistant cells. Furthermore, transcriptome analysis revealed that differentially expressed genes are functionally linked to a variety of biological functions that may lead to resistance to the inhibitors. These results may offer valuable insight into further understanding of BRAFi/MEKi resistance, as well as to the development of therapeutic tools to overcome drug resistance.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.

Integrated Genomics Identifies miR-181/TFAM Pathway as a Critical Driver of Drug Resistance in Melanoma

MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.

Unbiased High-Throughput Drug Combination Pilot Screening Identifies Synergistic Drug Combinations Effective against Patient-Derived and Drug-Resistant Melanoma Cell Lines.

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E-BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases.Pairwise DCs between dasatinib and three drugs approved for melanoma therapy-dabrafenib, vemurafenib, or trametinib-were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.

The positive correlation between drug addiction and drug dosage in vemurafenib-resistant melanoma cells is underpinned by activation of ERK1/2-FRA-1 pathway.

Malignant melanoma is a kind of highly invasive and deadly diseases. The BRAF inhibitor (BRAFi) such as vemurafenib could achieve a high response rate in melanoma patients with BRAF mutation. However, melanoma cells could easily develop resistance as well as addiction to BRAFi. Based on the drug addiction, intermittent treatment has been proposed to select against BRAFi-resistant melanoma cells. Because different dosages of BRAFi might be used in patients, it is necessary to know about the relationship between drug dosage and the degree of addiction. To address the problem, four drug-resistant melanoma cell sublines (A375/R0.5, A375/R2.0, M14/R0.5 and M14/R2.0) were established by continuously exposure of melanoma A375 or M14 cells to 0.5 or 2.0 μM vemurafenib. Vemurafenib withdrawal resulted in much stronger suppression on clone formation in A375/R2.0 and M14/R2.0, compared with A375/R0.5 and M14/R0.5, respectively. Meanwhile, stronger upregulation of ERK1/2-FRA-1 pathway could be observed in A375/R2.0 and M14/R2.0. Further detection showed that some proinflammatory cytokines downstream of ERK1/2-FRA-1 pathway were upregulated after drug withdrawal, and the conditioned medium collected from the resistant A375 cells could inhibit clone formation. Furthermore, vemurafenib withdrawal resulted in suppressed cell proliferation rather than cell senescence, with stronger effect on A375/R2.0 compared with A375/R0.5. This study suggested that the depth of vemurafenib addiction in resistant melanoma cells is positively correlated to the drug dosage, which might be underpinned by the ERK1/2-FRA-1 pathway and the related cytokines.

Autocrine Signaling of NRP1 Ligand Galectin-1 Elicits Resistance to BRAF-Targeted Therapy in Melanoma Cells.

Melanoma cells addicted to mutated BRAF oncogene activity can be targeted by specific kinase inhibitors until they develop resistance to therapy. We observed that the expression of Galectin-1 (Gal-1), a soluble ligand of Neuropilin-1 (NRP1), is upregulated in melanoma tumor samples and melanoma cells resistant to BRAF-targeted therapy. We then demonstrated that Gal-1 is a novel driver of resistance to BRAF inhibitors in melanoma and that its activity is linked to the concomitant upregulation of the NRP1 receptor observed in drug-resistant cells. Mechanistically, Gal-1 sustains increased expression of NRP1 and EGFR in drug-resistant melanoma cells. Moreover, consistent with its role as a NRP1 ligand, Gal-1 negatively controls p27 levels, a mechanism previously found to enable EGFR upregulation in cancer cells. Finally, the combined treatment with a Gal-1 inhibitor and a NRP1 blocking drug enabled resistant melanoma cell resensitization to BRAF-targeted therapy. In summary, we found that the activation of Galectin-1/NRP1 autocrine signaling is a new mechanism conferring independence from BRAF kinase activity to oncogene-addicted melanoma cells.

672 Targeting lncRNAs AC and BX reveals their critical role in melanoma and allows to identify novel phosphor-catalytic vulnerabilities

More than 80% percent of melanomas harbor mutations in either NRAS or BRAF. Our goal was to identify targets that are key in the process of melanocyte tumorigenesis and molecules that can be used to prevent this event. We have identified two long non-coding RNA (lncRNA) transcripts AC and BX key in the process of melanocyte tumorigenesis and melanoma progression. Tested in a variety of BRAF and NRAS mutated, drug resistant melanoma cell lines and other NRAS cancers such as lung ca and neuroblastoma, RNAi and antisense oligonucleotide (ASO) mediated knockdown of AC and BX lead to a vast reduction of tumor growth in vivo. High-throughput Kinase Activity Mapping (HT-KAM), a method for mapping phospho-catalytic dependencies of cells, unveiled AC and BX dependent kinases. Their inhibition not only mimic the knockdown-effects but also shows synergistic effect with ASO treatment. In addition, by using HT KAM technology, we were able to identify phospho-catalytic vulnerabilities of melanoma cells resistant to current targeted therapeutics. Moreover, knockdown of our transcripts causes a significant induction of PDL1 receptor ligand (key for immunotherapy response). Our studies are innovative because we offer new therapeutics and a diagnostic test to identify new drug combinations to restore therapeutic response for immune and targeted therapy.

Molecular alterations associated with acquired resistance to BRAFV600E targeted therapy in melanoma cells.

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.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Abstract Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3589.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC 50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3589.

P38 MAPK activation through B7-H3-mediated DUSP10 repression promotes chemoresistance

Immunoregulatory protein B7-H3 is involved in the oncogenic and metastatic potential of cancer cells, as well as in drug resistance. Resistance to conventional chemotherapy is an important aspect of melanoma treatment, and a better understanding of how B7-H3 enhances drug resistance may lead to the development of more effective therapies. We investigated the in vitro and in vivo sensitivity of chemotherapeutic agents dacarbazine (DTIC) and cisplatin in sensitive and drug resistant melanoma cells with knockdown expression of B7-H3. We found that knockdown of B7-H3 increased in vitro and in vivo sensitivity of melanoma cells to the chemotherapeutic agents dacarbazine (DTIC) and cisplatin, in parallel with a decrease in p38 MAPK phosphorylation. Importantly, in B7-H3 knockdown cells we observed an increase in the expression of dual-specific MAP kinase phosphatase (MKP) DUSP10, a MKP known to dephosphorylate and inactivate p38 MAPK. DUSP10 knockdown by siRNA resulted in a reversion of the increased DTIC-sensitivity seen in B7-H3 knockdown cells. Our findings highlight the potential therapeutic benefit of combining chemotherapy with B7-H3 inhibition, and indicate that B7-H3 mediated chemoresistance in melanoma cells is driven through a mechanism involving DUSP10-mediated inactivation of p38 MAPK.