Wild-type BRAF Melanoma Cells Research Articles

Abstract 4371: ERBB4 heterodimers appear to drive BRAFWT melanoma cell lines

Abstract Purpose of The Study: To determine whether heterodimerization of ERBB4 with EGFR or ERBB2 drives the proliferation of BRAF WT melanoma cell lines. Introduction: Metastatic melanomas that possess wild-type (WT) BRAF alleles are just as aggressive as melanomas that harbor gain-of-function (e.g., V600E) BRAF alleles. Moreover, no targets for effective chemotherapeutic intervention (other than immune checkpoint inhibitors) have been identified for these tumors. Thus, our goal is to identify candidate targets for therapeutic intervention in BRAF WT melanomas. ERBB4 (HER4) is a receptor tyrosine kinase that is closely related to the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). We have previously demonstrated that ERBB4 is both sufficient and necessary for the proliferation of BRAF WT melanoma cell lines. However, the constitutively homodimerized ERBB4 Q646C mutant inhibits the proliferation of BRAF WT melanoma cell lines. We have also previously demonstrated that ERBB4-EGFR and ERBB4-ERBB2 heterodimers stimulate proliferation and other oncogenic phenotypes in a variety of tumor model systems. Hence, here we test the hypothesis that ERBB4-EGFR or ERBB4-ERBB2 heterodimers drive BRAF WT melanomas. Experimental Approach: We are testing whether wild-type EGFR or ERBB2 alleles stimulate the proliferation of four different human BRAF WT melanoma cell lines. We are testing whether the dominant-negative EGFR (K721A) or ERBB2 (K753A) mutant alleles inhibit the proliferation of these cell lines. Likewise, we are testing whether EGFR or ERBB2 shRNAs inhibit the proliferation of these cell lines. Finally, we are testing whether EGFR or ERBB2 enables ERBB4 mutant alleles found in BRAF WT melanoma samples to drive greater levels of proliferation in 32D cells than wild-type ERBB4. Unpublished Results: Wild-type ERBB2 causes a significant increase in the proliferation of the MEL-JUSO and IPC-298 BRAF WT female melanoma cell lines. The ERBB2 dominant-negative allele causes a significant decrease in the proliferation of these cell lines. Finally, preliminary data suggest that the EGFR and ERBB2 dominant-negative alleles inhibit the proliferation of the MeWo BRAF WT male melanoma cell line. Preliminary Conclusions: Our data suggest that ERBB2-ERBB4 heterodimers drive two female BRAF WT melanoma cell lines, whereas ERBB2-ERBB4 and EGFR-ERBB4 heterodimers drive a male BRAF WT melanoma cell line. Thus, we predict that FDA-approved ERBB2 inhibitors (and in some cases, EGFR inhibitors) may be effective in ERBB4-dependent, BRAF WT melanomas. Moreover, ERBB4 heterodimerization with ERBB2 or EGFR may account for the gender disparities observed in BRAF WT melanomas. Citation Format: Vipasha Dwivedi, Lauren M. Lucas, Jen Davis, Kaitlyn O'Daniel, Rees H. Cooke, Joelle N. Woggerman, Ella N. Wilson, Maddy N. Ingrao, David J. Riese. ERBB4 heterodimers appear to drive BRAFWT melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4371.

MiR-224–5p acts as a tumour suppressor and reverses the resistance to BRAF inhibitor in melanoma through directly targeting PAK4 to block the MAPK pathway

miR-224–5p has been shown to play both an oncogene and tumour suppressor role in many human tumours. However, the role and molecular mechanism of miR-224–5p in cutaneous melanoma remains unclear. miR-224–5p levels were downregulated in melanoma tissue, and low miR-224–5p expression was an independent risk factor for melanoma patients. miR-224–5p blocked proliferation, epithelial-to-mesenchymal transition (EMT), invasion, migration in BRAF wild-type melanoma cell, and overcome acquired BRAFi resistance in VMF-resistant melanoma cells. miR-224–5p exerted its role by directly repressing PAK4 to block the downstream CRAF/MEK/ERK pathways. We demonstrated that miR-224–5p inhibited melanoma growth and metastasis in vivo though xenograft tumor and pulmonary metastasis assay. Thus, miR-224–5p/PAK4-mediated CRAF/MEK/ERK pathways have therapeutic potential in melanoma treatment.

Prognostic and predictive value of metformin in the European Organisation for Research and Treatment of Cancer 1325/KEYNOTE-054 phase III trial of pembrolizumab versus placebo in resected high-risk stage III melanoma

BackgroundMetformin is a commonly prescribed and well-tolerated medication. In laboratory studies, metformin suppresses BRAF wild-type melanoma cells but accelerates the growth of BRAF-mutated cells. This study investigated the prognostic and predictive value of metformin, including with respect to BRAF mutation status, in the European Organisation for Research and Treatment of Cancer 1325/KEYNOTE-054 randomised controlled trial. MethodsPatients with resected high-risk stage IIIA, IIIB, or IIIC melanoma received 200 mg of pembrolizumab (n = 514) or placebo (n = 505) every 3 weeks for twelve months. Pembrolizumab prolonged recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) at approximately 42 months median follow-up (Eggermont et al., TLO, 2021). Multivariable Cox regression was used to estimate associations of metformin with RFS and DMFS. Interaction terms were used to model effect modification by treatment and BRAF mutation. ResultsFifty-four patients (0.5%) used metformin at baseline. Metformin was not significantly associated with RFS (hazard ratio [HR] 0.87, 95% confidence interval [CI] 0.52–1.45) and DMFS (HR 0.82, 95% CI 0.47–1.44). The interaction between metformin and the treatment arm was not significant for either RFS (p = 0.92) or DMFS (p = 0.93). Among patients with mutated BRAF, the association of metformin with RFS (HR 0.70, 95% CI 0.37–1.33) was greater in magnitude though not significantly different to those without mutated BRAF (HR 0.98, 95% CI 0.56–1.69). ConclusionsThere was no significant impact of metformin use on pembrolizumab efficacy in resected high-risk stage III melanoma. However, larger studies or pooled analyses are needed, particularly to explore a possible effect of metformin in BRAF-mutated melanoma.

Cytotoxicity, acute and sub-chronic toxicities of the fruit extract of Tetrapleura tetraptera (Schumm. & Thonn.) Taub. (Fabaceae)

BackgroundTetrapleura tetraptera is a medicinal spice traditionally used to treat cancer, diabetes, and several other ailments. This study analyzed the cytotoxicity of the dichloromethane methanol extract of T. tetraptera fruits (TTF) and its constituents. The toxicity profile of the TTF extract was also evaluated in rats.MethodsThe Cytotoxicity of this extract was evaluated using the resazurin reduction assay (RRA). Acute and sub-chronic toxicity studies were performed according to the protocol described by the Organisation for Economic Cooperation, and Development (OECD). Hematological, serum, and urine biochemical parameters, as well as histological sections of the liver and kidney, were also evaluated based on standard methods.ResultsThe TTF extract, compound 5, and the reference drug doxorubicin were active in all 9 tested cancer cell lines. The recorded IC50 ranged from 18.32 μM (against B16-F1 murine melanoma cells) to 36.18 μM (against SKMel-505 BRAF wildtype melanoma cells) for TTF, from 10.02 μM (towards MaMel-80a BRAF-V600E homozygous mutant melanoma cells) to 31.73 μM (against SKMel-28 BRAF-V600E homozygous mutant melanoma cells) for compound 5, and from 0.22 μM (against B16-F1 cells) to 9.39 μM (against SKMel-505 cells) for doxorubicin. The study of acute toxicity test showed that the lethal dose (LD50) of this extract was greater than 5000 mg/kg body weight. In the sub-chronic toxicity studies, variations were observed in some biochemical parameters, especially at higher doses.ConclusionTTF and its most active compound (5) are found to be potential cytotoxic agents, meanwhile, TTF was safe when given a single oral dose of 5000 mg/kg. However, caution is necessary in case of prolonged oral administration due to potential alterations of renal function at high doses (> 1000 mg/kg).

Abstract 4000: ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines

Abstract Approximately 50% of metastatic melanomas possess wild-type (WT) BRAF alleles. These BRAF-WT tumors are a significant clinical problem, despite the fact that these tumors commonly possess mutations in NF1 or RAS genes, as there are no targeted therapeutics for these tumors. Here we demonstrate that ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines, suggesting that disrupting ERBB4 signaling may be effective against BRAF WT melanomas. ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are validated targets in a variety of solid tumors, whereas the roles that ERBB4 plays in human malignancies are ambiguous. Our previous in silico analyses of BRAF-WT tumor genomes in The Cancer Genome Atlas skin cutaneous melanoma (TCGA-SKCM) dataset suggest that ERBB4 mutation or overexpression cooperates with NF1 or RAS gene mutations to drive melanomas via activation of the PI3 kinase/AKT signaling pathway. These in silico analyses have also enabled us to prioritize the ERBB4 mutants found in BRAF-WT melanomas. We have tested this hypothesis using a panel of human BRAF-WT melanoma cell lines. We have shown that expression of wild-type ERBB4 causes increased clonogenic proliferation, whereas expression of a dominant-negative ERBB4 mutant (K751M) causes decreased clonogenic proliferation. These data indicate that ERBB4 is sufficient and necessary for clonogenic proliferation of BRAF-WT melanoma cell lines. Analgous studies are testing whether ERBB4 regulates anchorage independence in semi-solid medium and proliferation rates and saturation density in culture dishes. The BRAF-WT melanoma cell lines endogenously express ERBB4 ligands, EGFR, and ERBB2. Moreover, ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR and ERBB4-ERBB2 heterodimers are oncogenic. Thus, we are using gene silencing and pharmacologic tools to determine whether endogenous ERBB4 ligands, endogenous EGFR, or endogenous ERBB2 is required for the oncogenic activity of ERBB4. Furthermore, we are determining whether prioritized ERBB4 mutants found in BRAF-WT melanomas exhibit greater signaling and oncogenic activities than WT ERBB4. Consequently, these experiments will establish a functional role for ERBB4 mutations and expression in BRAF-WT melanomas and will identify potential strategies for disrupting oncogenic ERBB4 signaling in these tumors. Citation Format: Lauren M. Lucas, Vipasha Dwivedi, Rania H. Mohamedelhassan, Erin L. Harrell, Connor M. Kelley, Elizabeth L. Knerr, Jessica A. Markham, David J. Riese. ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines [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 4000.

Botanical from the Fruits Mesocarp of Raphia vinifera Displays Antiproliferative Activity and Is Harmless as Evidenced by Toxicological Assessments.

Raphia vinifera is widely used to treat several diseases including digestive disorders, dysentery, and genitourinary infections. In this study, the mineral contents, the cytotoxicity, and the toxicological effect of the crude CHCl3/MeOH extract (RVM) from the mesocarp of Raphia vinifera were evaluated. The mineral contents were evaluated using the method described by the Association of Official Analytical Chemists (AOAC). The cytotoxicity of both extract and chemical compounds from the plants was determined by a resazurin reduction assay (RRA). The toxicological studies were carried out using the experimental procedure of the Organization for Economic Cooperation and Development (OECD). After killing the rats, biochemical, histopathological, and hematological studies were performed. The result indicated that RVM is rich in zinc (6.52 mg/100 g of DM) and sodium (194.5 mg/100 g of DM). RVM had a cytotoxicity effect with IC50 values lower than 30 μg/mL in 18/18 cancer cell lines tested. These recorded IC50 values were between 12.35 µg/mL (toward CCRF-CEM leukemia cells) and 26.66 µg/mL (toward SKMel-505 BRAF wild-type melanoma cells). Raphvinin 4 displayed good cytotoxicity against MaMel-80aBRAF-V600E homozygous mutant with the IC50 of 10.42 μM. RVM was relatively nontoxic to rats, the median lethal dose (DL50) being above 5000 mg/kg body weight. However, during the oral administration period extending for 28 days, precautions should be taken due to the increase in urinary creatinine level and decrease in spleen weight in the male rats given the highest dose (1000 mg/kg) of extract. Conclusively, the extract of Raphia vinifera is weakly toxic in rats and could be further used in the development of anticancer phytomedicines.

GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.

Repurposing the serotonin agonist Tegaserod as an anticancer agent in melanoma: molecular mechanisms and clinical implications

BackgroundNew therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors.MethodsDrug screening, IC50 determinations as well as synergy assays were detected by the MTT assay. Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry. TUNEL staining was performed using immunocytochemistry. Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry. To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used. Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors. Evaluation of immune infiltrates was carried out by flow cytometry.ResultsUsing a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines. Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS). TM’s anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition. Specifically, TM blunted S6 phosphorylation in both BRAFV600E and BRAF wildtype (WT) melanoma cell lines. TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model. In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation. Furthermore TM decreased the infiltration of immune suppressive regulatory CD4+CD25+ T cells and FOXP3 and ROR-γt positive CD4+ T cells. Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAFV600E mutation and could be additively or synergistically combined with Cobimetinib in both BRAFV600E and BRAF WT melanoma cell lines in inducing anti-cancer effects.ConclusionTaken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAFV600E and BRAF WT melanoma.

Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma.

Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAFi) and histone deacetylase inhibitors (HDACi) to enhance the delivery of MC1R-targeted radiolabeled peptide ([212Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAFV600E cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [212Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAFi and/or HDACi was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAFi and HDACi significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAFV600E and BRAFWT cells. Combining [212Pb]DOTA-MC1L with BRAFi and/or HDACi improved the tumor response by increasing the delivery of 212Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDACi and BRAFi could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.

Critical role of miR-10b in B-RafV600E dependent anchorage independent growth and invasion of melanoma cells.

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the B-Raf genetic locus as one of the critical events in melanomagenesis. B-Raf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of B-Raf is tightly regulated and is required for cell growth and survival. B-Raf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of B-Raf mutated melanoma cells are stringently dependent on B-Raf-MEK-ERK activation, the downstream effector targets that are required for oncogenic B-Raf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic B-Raf (B-RafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of B-Raf wild-type melanoma cells, miR-10b silencing decreases B-RafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for B-RafV600E-mediated anchorage independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic B-RafV600E activity in melanoma.

Abstract 5419: The relationship between glutamatergic signaling and altered glutamine metabolism in melanoma

Abstract Aberrant glutamatergic signaling has been implicated in many cancer types, and is associated with dysregulated growth leading to cellular transformation and tumorigenesis. Our laboratory has previously illustrated the oncogenic properties of a neuronal receptor, metabotropic glutamate receptor 1 (GRM1) in melanocytes. Glutamate is the natural ligand of GRM1 and the major excitatory neurotransmitter in the central nervous system. Our group has demonstrated that glutamate production/release is upregulated in GRM1 expressing melanoma cells, resulting in constitutive activation of GRM1 and GRM1-associated downstream signaling pathways. We hypothesize that this activation of GRM1 in melanoma cells is associated with higher expression of c-Myc and increased enzymatic activity of glutaminase (GLS) converting glutamine to glutamate. We showed that reducing the extracellular glutamate levels by an inhibitor of glutamate release, Riluzole, led to significantly reduce melanoma cell proliferation in vitro and tumor progression in vivo. We also demonstrated that inclusion of CB-839, a potent, selective, and orally bioavailable GLS inhibitor, resulted in a significant inhibition of GRM1-expressing melanoma cell proliferation compared to GRM1 negative melanoma cell lines. Interestingly, we found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1 expressing melanoma cells led to enhanced suppression of cell proliferation in vitro. Additionally, we found that wild type BRAF melanoma cells are more sensitive to CB-839 than mutated BRAF cells while responses to Riluzole were independent of BRAF genotypes. Furthermore, our data indicate that CB-839 acts by reducing the function, not the expression, of GLS. Currently, we are investigating the relationship between GRM1, c-Myc and GLS by genetic and pharmacological approaches, and if this cascade of events is linked with altered glutamine metabolisms described for many human cancers including melanoma. Citation Format: Raj Shah, Andrew Boreland, Suzie Chen. The relationship between glutamatergic signaling and altered glutamine metabolism in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5419. doi:10.1158/1538-7445.AM2017-5419

The influence of aranoza drug formulations and «empty» liposomes on the expression of PD-L1 и PD-L2 in human melanoma cell lines

Introduction. In recent years, significant progress has been achieved in immunotherapy of tumors, however, little is known about the crosstalk between the tumor and the immune system. It seems reasonable to rise the question of patients treatment with PD-1 and its ligands blockers after chemotherapy. Objective. To investigate the expression of cell surface molecules PD-L1 and PD-L2 both at the mRNA and protein levels in human melanoma cell lines after exposure to «Aranoza, lyophilisate for preparation of solution for injections» (aranoza-lio), liposomal formulation of aranoza and «empty» liposomes without aranoza. Materials and methods. In this study we used 11 melanoma cell lines, 5 of which carried the BRAF mutation. In quantitative polymerase chain reaction in real time we investigated the level of PD-L1 and PD-L2 gene expression. Using flow cytometry we evaluated the expression of cell surface antigens PD-L1 and PD-L2. Results. PD-L1 and PD-L2 mRNA are expressed at a lower level in cells with BRAF mutations, differences were significant only for PD-L2 (p = 0.1373 andp = 0.0207respectively). A high basal level of protein PD-L1 and PD-L2 expression was observed in wild type BRAF melanoma cells. After exposure to liposomal aranoza the level of PD-L1 and PDL-2 mRNA expression was significantly reduced (p = 0.0004 and p = 0.0442 respectively) in compare to control non-treated cells. It is of interest, aranoza-lio and «empty» liposomes increased the expression of PD-L1 (p <0.0001 and p = 0.0005 respectively) and PDL2 (p = 0.0005 and p = 0.0025). Of note, we observed an opposite effect in some melanoma cell lines. Moreover, changes in the expression of proteins PD-L1 and PD-L2 did not correlate with the mRNA level. The expression of PD-L1 protein after incubation with the liposomal aranoza increased dramatically compared to non-treated cells (p = 0.0269). Aranoza-lio and «empty» liposomes decreased the expression of PD-L1 protein (p = 0.0663 and p = 0.7213 respectively). The protein level of PD-L2 after exposure to liposomal aranoza did not change significantly (p = 0.1141), however, aranoza-lio and «empty» liposomes reduced the expression of PD-L2 (p = 0.0021 andp = 0.008). Conclusion. These findings are consistent with those of other researchers and confirm that the level of PD-L1 and PD-L2 mRNA and protein are modulated after treatment. Also, various drug formulations of Aranoza, as well as the «empty» liposomes, have different effects on the expression of PD-L1 and PD-L2 mRNA and proteins.

Targeting CPT1A enhances metabolic therapy in human melanoma cells with the BRAF V600E mutation

Cancer cells are characterized by altered metabolic processes. Recent evidence of metabolic alterations has indicated that the fatty acid oxidation (FAO) pathway is used as a carbon source for anabolic processes in some tumors, thus making this pathway a potential target for therapy. The carnitine palmitoyltransferase (CPT; EC 2.3.1.21) enzyme transfers long-chain fatty acids from the cytosol to the mitochondrial matrix for β-oxidation. Because carnitine palmitoyl transferase 1a (CPT1a) is the rate-limiting enzyme for FAO, the authors evaluated the effects of CPT1A knock-down in BRAF V600E melanoma cell lines. The results showed that knock-down of CPT1A inhibited FAO and that CPT1A is critical for malignant V600E melanoma cells, particularly BRAF V600E melanoma cells. The proliferation and tumorigenesis in V600E melanoma were decrease after CPT1A knockdown. These results suggest that therapy for BRAF V600E melanoma can include targeting metabolic alterations. CPT1A is more important for lipid synthesis in V600E mutant melanoma cells than in wild-type BRAF melanoma cells.

A novel compound which sensitizes BRAF wild-type melanoma cells to vemurafenib in a TRIM16-dependent manner

There is an urgent need for better therapeutic options for advanced melanoma patients, particularly those without the BRAFV600E/K mutation. In melanoma cells, loss of TRIM16 expression is a marker of cell migration and metastasis, while the BRAF inhibitor, vemurafenib, induces melanoma cell growth arrest in a TRIM16-dependent manner. Here we identify a novel small molecule compound which sensitized BRAF wild-type melanoma cells to vemurafenib. High throughput, cell-based, chemical library screening identified a compound (C012) which significantly reduced melanoma cell viability, with limited toxicity for normal human fibroblasts. When combined with the BRAFV600E/K inhibitor, vemurafenib, C012 synergistically increased vemurafenib potency in 5 BRAFWT and 4 out of 5 BRAFV600E human melanoma cell lines (Combination Index: CI < 1), and, dramatically reduced colony forming ability. In addition, this drug combination was significantly anti-tumorigenic in vivo in a melanoma xenograft mouse model. The combination of vemurafenib and C012 markedly increased expression of TRIM16 protein, and knockdown of TRIM16 significantly reduced the growth inhibitory effects of the vemurafenib and C012 combination. These findings suggest that the combination of C012 and vemurafenib may have therapeutic potential for the treatment of melanoma, and, that reactivation of TRIM16 may be an effective strategy for patients with this disease.

Abstract 1883: Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells

Abstract The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4b expression in gastric, colon and breast cancer cells. In the present study we treated BRAF mutant and BRAF wild type melanoma cells with HDAC inhibitors (SAHA (suberanilohydroxamic acid) or valproic acid) and tested the changes in abundance, localization and function of PMCA4b. Expression levels of the PMCA proteins were analyzed by qRT-PCR and Western Blotting. Subcellular localization of the PMCAs and the effects of treatments on cytosolic Ca2+ signaling were analyzed by confocal microscopy. We found that treatment with the HDAC inhibitors increased the level of PMCA4b expression at both mRNA and protein levels in both BRAF wild type and BRAF mutant cell types. The increased PMCA4b level was coupled with enhanced plasma membrane localization and with a faster Ca2+ clearance after stimulation. Our results show that in melanoma cells the expression of PMCA4b is under epigenetic control and HDAC inhibitors efficiently enhanced PMCA4b expression independent of the BRAF status of cells. Citation Format: Luca Hegedus, Tamas Garay, Eszter Molnar, Karolina Varga, Rita Padanyi, Katalin Paszty, Balazs Hegedus, Mathias Wolf, Michael Grusch, Eniko Kallay, Agnes Enyedi. Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1883.

Repression of microRNA-768-3p by MEK/ERK signalling contributes to enhanced mRNA translation in human melanoma.

Increased global protein synthesis and selective translation of mRNAs encoding proteins contributing to malignancy is common in cancer cells. This is often associated with elevated expression of eukaryotic translation initiation factor 4 (eIF4E), the rate-limiting factor of cap-dependent translation initiation. We report here that in human melanoma downregulation of miR-768-3p as a result of activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway has an important role in the upregulation of eIF4E and enhancement in protein synthesis. Melanoma cells displayed increased nascent protein production and elevated eIF4E expression, which was associated with the downregulation of miR-768-3p that was predicted to target the 3'-untranslated region of the eIF4E mRNA. Overexpression of miR-768-3p led to the downregulation of the endogenous eIF4E protein, reduction in nascent protein synthesis and inhibition of cell survival and proliferation. These effects were efficiently reversed when eIF4E was co-overexpressed in melanoma cells. On the other hand, introduction of anti-miR-768-3p into melanocytes upregulated endogenous eIF4E protein expression and increased global protein synthesis. Downregulation of miR-768-3p appeared to be mediated by activation of the MEK/ERK pathway, in that treatment of BRAF(V600E) melanoma cells with the mutant BRAF inhibitor PLX4720 or exposure of either BRAF(V600E) or wild-type BRAF melanoma cells to the MEK inhibitor U0126 resulted in the upregulation of miR-768-3p and inhibition of nascent protein synthesis. This inhibition was partially blocked in cells cointroduced with anti-miR-768-3p. Significantly, miR-768-3p was similarly downregulated, which was inversely associated with the expression levels of eIF4E in fresh melanoma isolates. Taken together, these results identify downregulation of miR-768-3p and subsequent upregulation of eIF4E as an important mechanism in addition to phosphorylation of eIF4E responsible for MEK/ERK-mediated enhancement of protein synthesis in melanoma.

Selective Targeting of CTNBB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs.

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for β-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.

Combined inhibition of MEK and mTOR has a synergic effect on angiosarcoma tumorgrafts.

Angiosarcoma (AS) is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. Using tumorgraft models, we previously showed that AS is sensitive to small-molecule inhibitors that target mitogen-activated/extracellular-signal-regulated protein kinase kinases 1 and 2 (MEK). The objective of this study was to identify drugs that combine with MEK inhibitors to more effectively inhibit AS growth. We examined the in vitro synergy between the MEK inhibitor PD0325901 and inhibitors of eleven common cancer pathways in melanoma cell lines and canine angiosarcoma cell isolates. Combination indices were calculated using the Chou-Talalay method. Optimized combination therapies were evaluated in vivo for toxicity and efficacy using canine angiosarcoma tumorgrafts. Among the drugs we tested, rapamycin stood out because it showed strong synergy with PD0325901 at nanomolar concentrations. We observed that angiosarcomas are insensitive to mTOR inhibition. However, treatment with nanomolar levels of mTOR inhibitor renders these cells as sensitive to MEK inhibition as a melanoma cell line with mutant BRAF. Similar results were observed in B-Raf wild-type melanoma cells as well as in vivo, where treatment of canine AS tumorgrafts with MEK and mTOR inhibitors was more effective than monotherapy. Our data show that a low dose of an mTOR inhibitor can dramatically enhance angiosarcoma and melanoma response to MEK inhibition, potentially widening the field of applications for MEK-targeted therapy.

P8.05 - Selective targeting of CTNBB1-, KRAS- or MYC-driven cell growth by combinations of existing drugs

ABSTRACT The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for b-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in CTNNB1-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.

Differential effects of the oncogenic BRAF inhibitor PLX4032 (vemurafenib) and its progenitor PLX4720 on ABCB1 function.

The clinically approved oncogenic BRAF inhibitor PLX4032 (vemurafenib) was shown to be a substrate of the ATP-binding cassette (ABC) transporter ABCB1. Here, we compared PLX4032 and its structurally closely related precursor compound PLX4720 for their interference with ABCB1 and the ABCB1-mediated compound transport using docking and cell culture experiments. For the docking study of PLX4032 and PLX4720 with ABCB1, we analysed binding of both compounds to mouse Abcb1a and to human ABCB1 using a homology model of human ABCB1 based on the 3D structure of Abcb1a. Naturally ABCB1 expressing cells including V600E BRAF-mutated and BRAF wild-type melanoma cells and cells transduced with a lentiviral vector encoding for ABCB1 were used as cell culture models. ABCB1 expression and function were studied by the use of fluorescent and cytotoxic ABCB1 substrates in combination with ABCB1 inhibitors. Docking experiments predicted PLX4032 to interact stronger with ABCB1 than PLX4720. Experimental studies using different cellular models and structurally different ABCB1 substrates confirmed that PLX4032 interfered stronger with ABCB1 function than PLX4720. For example, PLX4032 (20 µM) induced a 4-fold enhanced rhodamine 123 accumulation compared to PLX4720 (20 µM) in ABCB1-transduced UKF-NB-3 cells and reduced the IC₅₀ for the cytotoxic ABCB1 substrate vincristine in this model by 21-fold in contrast to a 9-fold decrease induced by PLX4720. PLX4032 exerted stronger effects on ABCB1-mediated drug transport than PLX4720. This indicates that small changes in a molecule can substantially modify its interaction with ABCB1, a promiscuous transporter that transports structurally different compounds.