Panel Of Melanoma Cell Lines Research Articles

P21-Activated Kinase 1 (PAK1) as a Therapeutic Target in BRAF Wild-Type Melanoma

Although remarkable clinical response rates in melanoma have been observed using vemurafenib or dabrafenib in patients with tumors carrying oncogenic mutations in BRAF, a substantial unmet medical need remains for the subset of patients with wild-type BRAF tumors. To investigate the role of p21-activated kinases (PAKs) in melanoma, we determined PAK1 genomic copy number and protein expression for a panel of human melanoma tissues. PAK1 was inhibited in vitro and in vivo using RNA interference or PF-3758309 inhibitor treatment in a panel of melanoma cell lines with known BRAF and RAS (rat sarcoma) genotype to better understand its role in melanoma cell proliferation and migration. Tumorigenesis was assessed in vivo in female NCR nude mice and analyzed with cubic spline regression and area under the curve analyses. All statistical tests were two-sided. Strong cytoplasmic PAK1 protein expression was prevalent in melanomas (27%) and negatively associated with activating mutation of the BRAF oncogene (P < .001). Focal copy number gain of PAK1 at 11q13 was also observed in 9% of melanomas (n = 87; copy number ≥ 2.5) and was mutually exclusive with BRAF mutation (P < .005). Selective PAK1 inhibition attenuated signaling through mitogen-activated protein kinase (MAPK) as well as cytoskeleton-regulating pathways to modulate the proliferation and migration of BRAF wild-type melanoma cells. Treatment of BRAF wild-type melanomas with PF-3758309 PAK inhibitor decreased tumor growth for SK-MEL23 and 537MEL xenografts (91% and 63% inhibition, respectively; P < .001) and MAPK pathway activation in vivo. Taken together, our results provide evidence for a functional role of PAK1 in BRAF wild-type melanoma and therapeutic use of PAK inhibitors in this indication.

The Anti-Melanoma Activity of Dinaciclib, a Cyclin-Dependent Kinase Inhibitor, Is Dependent on p53 Signaling

Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.

Variation in susceptibility of human malignant melanomas to oncolytic vesicular stomatitis virus

Vesicular stomatitis virus (VSV) is a novel, anti-cancer therapy that targets cancer cells selectively with defective antiviral responses; however, not all malignant cells are sensitive to the oncolytic effects of VSV. Herein, we have explored the mechanistic determinants of mutant M protein VSV (M51R-VSV) susceptibility in malignant melanoma cells. Cell viability after VSV infection was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assay in a panel of melanoma cell lines. VSV infectability, viral protein synthesis, and viral progeny production were quantified by flow cytometry, (35)S-methionine electrophoresis, and viral plaque assays, respectively. Interferon (IFN) responsiveness was determined using MTS assay after β-IFN pretreatment. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV. Cell viability after M51R-VSV infection at a multiplicity of infection of 10 pfu/mL, 48 hours postinfection) ranged between 0 ± 1% and 59 ± 9% (mean ± standard deviation). Sensitive cell lines supported VSV infection, viral protein synthesis, and viral progeny production. In addition, when pretreated with β-IFN, sensitive cells became resistant to M51R-VSV, suggesting that IFN-mediated antiviral signaling is defective in these cells. In contrast, resistant melanoma cells do not support VSV infection, viral protein synthesis, or viral replication, indicating that antiviral defenses remain intact. In a murine xenograft model, intratumoral M51R-VSV treatment decreased tumor growth relative to controls after 26 days in SK-Mel 5 (-21 ± 19% vs. 2,100 ± 770%; P < .0001) and in SK-Mel 3 (2,000 ± 810% vs. 7,000 ± 3,000%; P = .008) established tumors. M51R-VSV is a viable anti-cancer therapy, but susceptibility varies among melanomas. Future work will exploit specific mechanisms of resistance to expand the therapeutic efficacy of M51R-VSV.

Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

BackgroundMelanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines.ResultsThe majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1.ConclusionsOur data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Effect of Culture at Low Oxygen Tension on the Expression of Heat Shock Proteins in a Panel of Melanoma Cell Lines

ObjectiveTumours are commonly hypoxic and this can be associated with aggressive tumour type, metastasis and resistance to therapy. Heat shock proteins (hsps) are induced in response to hypoxia, provide cancer cells with protection against tumour-associated stressors and chaperone oncoproteins that drive tumour proliferation. This study examined the effect of different oxygen concentrations on the expression of hsps in melanoma cell lines.MethodsMelanoma cell lines were cultured in 2% and 20% O2. Expression of Hsp90, Hsp70, Hsp60, Hsp40 and Hsp32 proteins were determined by flow cytometry.ResultsGrowth rates and viability were reduced in the majority of cell lines by culture in 2% O2. Hsp expression was different in 2% compared to 20% O2 and changes in Hsp90 expression correlated with cell line generation time (P<0.005) and viability (P<0.01). Greater total hsp expression correlated with improved viability in 2% but not 20% O2 (P<0.05). Relative expression of the different hsps was consistent across cell lines and each correlated with the others (P = 0.0001) but not with Hsp32. Hsp expression was inversely correlated with cell line adhesion to laminin as well as collagen type IV and Breslow depth of the original primary tumour tissue (P<0.05), but not with Clark level or patient survival. All five hsps were identified on the cell surface.ConclusionCulture in 2% O2 variably altered hsp expression in a panel of melanoma cell lines. Hsp expression was associated with certain cell line characteristics and clinical parameters of the originating tumour.

General Sensitization of Melanoma Cells for TRAIL-Induced Apoptosis by the Potassium Channel Inhibitor TRAM-34 Depends on Release of SMAC

The death ligand TRAIL represents a promising therapeutic strategy for metastatic melanoma, however prevalent and inducible resistance limit its applicability. A new approach is presented here for sensitization to TRAIL. It is based on inhibition of the membrane potassium channel KCa3.1 (IK1), which serves fundamental cellular functions related to membrane potential. The selective inhibitor TRAM-34 did not induce apoptosis by itself but synergistically enhanced TRAIL sensitivity and overrode TRAIL resistance in a large panel of melanoma cell lines. Expression of IK1 was also found in mitochondria, and its inhibition resulted in mitochondrial membrane hyperpolarization and an early activation of Bax. The combination of TRAM-34 and TRAIL resulted in massive release of mitochondrial factors, cytochrome c, AIF and SMAC/DIABLO. Bax knockdown and Bcl-2 overexpression abolished apoptosis. Overexpression of XIAP diminished apoptosis by two-fold, and SMAC knockdown almost completely abolished apoptosis. These data uncover the existence of a rheostat in melanoma cells, consisting of inhibitor of apoptosis proteins and SMAC, which regulates TRAIL sensitivity. Thus, a new strategy is described based on mitochondrial membrane channels, which correspond to Bax activation. As both TRAIL and IK1 inhibitors had shown only minor side effects in clinical trials, a clinical application of this combination is conceivable.

Clinical significance of genomic alterations of the CDK4-pathway and sensitivity to the CDK4 inhibitor PD 0332991 in melanoma.

8520 Background: Activation of CDK4 by amplification, increased expression of Cyclin D1 (CCND1) or reduced expression of the CDK inhibitor p16 (CDKN2A) can contribute to transformation of melanocytes indicating that CDK4 can act as an oncogene in melanoma.To explore if CDK4 may be a viable target for the treatment of human melanoma we have analyzed the frequency and clinico-pathological associations of genomic alterations of the CDK4 pathway in primary human melanoma and examined the genomic predictors of sensitivity to the highly selective CDK4/6 inhibitor PD 0332991 (991) in a panel of melanoma cell lines. Methods: A series of 167 primary melanomas with clinical, molecular and pathological annotation, including median follow up of 6.6 years, were analyzed for copy number variation (CNV)- gain of CDK4 or CCND1 (average gene copy &gt;2.4) or loss of CDKN2A (average gene copy &lt;1.4), by fluorescence in situ hybridization. A panel of 39 cell lines were treated with 991 in vitro and GI50s calculated. The mean GI50 of the melanoma cell lines was used to define sensitivity. Gene expression profiling and mutation or CNV in CDKN2A were used to identify predictors of sensitivity. Results: 75% of primary melanomas had at least one CNV (75%, 70% and 82% for BRAF, NRAS or wild-type BRAF/NRAS mutation status respectively). 55% showed loss of CDKN2A. 28% of melanomas had two or more CNVs. Melanomas with two or more CNVs involving CCND1 had worse overall survival (HR 5.56, p=0.02). Low CDKN2A mRNA expression or mutation or loss of CDKN2A predicted sensitivity to 991 with 30/33 mutant/loss lines being sensitive compared to only 2/6 wild type lines (p&lt;0.006). Expression of CDK4, CCND1 or other cyclins or CDK-inhibitors did not predict sensitivity to 991. Conclusions: Genomic alterations in the CDK4 pathway are frequent in melanoma and are associated with worse survival, particularly when melanomas harbor two or more CNVs involving CCND1. Mutation, loss or low expression of CDKN2A in melanoma cell lines predicted sensitivity to the CDK4 inhibitor 991. Taken together these data support evaluation of CDK4 inhibitors in melanoma and suggest that CDKN2A maybe a genomic predictor of sensitivity to these agents.

Molecular profiling and comparative genomic hybridization analysis in human melanoma cell lines and identification of BRAF amplification in a PLX4032 acquired resistance cell line.

8592 Background: Melanoma is the most aggressive form of skin cancer. Its management is evolving rapidly due to an improved understanding of the molecular heterogeneity and the development of effective, personalized targeted therapies. PLX4032 is a BRAF inhibitor that induces tumor response in patients with BRAF mutation whose response is limited due to acquired resistance. We used comprehensive microarray and genomic analysis to molecularly characterize a panel of melanoma cell lines with the goal of identifying new molecular subgroups. To better understand the mechanisms of acquired resistance to PLX4032, we included two cell lines that were conditioned in vitro to acquire resistance. Methods: Using microarray, we studied 52 melanoma cell lines for mRNA expression and performed array CGH using genome microarrays. Data analysis was done using Rosetta Resolver and Agilent Analytics 4.0 software. Results: Microarray analysis suggests melanoma is comprised of at least two distinct molecular groups. One group has a differentiated melanocyte phenotype and the other has an expression signature characteristic of progenitor-like cells. The progenitor-like group expresses SOX9, WNT5A and WNT5B and also has the lowest relative expression of MITF. The most differentiated group had the highest MITF and SOX5 levels, while the progenitor-like cell lines have decreased expression. MITF appears to be a strong candidate marker for this group. Positive correlation exists between MITF expression levels and gene copy number, as almost all of the MITF samples amplified by CHG analysis are also overexpressed. A third group shows strong expression of SOX5, SOX10 and Nestin. One of the most important findings is M14-R cell line, conditioned to acquire PLX4032 resistance, has a focal, high level amplification of BRAF (Log2 ratio = 3, 8-fold amplification) when compared with the parental cell line, which is extremely sensitive to PLX4032. Conclusions: These results afford new insight into the potential pathogenesis and classification and provide a greater understanding of melanoma. BRAF amplification could be a novel mechanism of resistance to PLX4032.

Design, synthesis, and antiproliferative activity of new 1H-pyrrolo[3,2-c]pyridine derivatives against melanoma cell lines. Part 2

A new series of diarylureas and diarylamides possessing 1H-pyrrolo[3,2-c]pyridine scaffold was designed and synthesized. Their in vitro antiproliferative activities against A375P human melanoma cell line and NCI-9 human melanoma cell line panel were tested. All the target compounds, except three amino derivatives 8g, h and 9h, demonstrated superior potencies against A375P to Sorafenib. In addition, compounds 8a and 9b–f demonstrated higher potencies than Vemurafenib against A375P. Compounds 8c and 9b were 7.50 and 454.90 times, respectively, more selective towards A375P melanoma cells over NIH3T3 fibroblasts. Furthermore, compounds 8d, e and 9a–d, f demonstrated very high potencies against the nine tested melanoma cell lines at the NCI. The bisamide derivatives 9a–c, f showed 2-digit nanomolar IC50 values over different cell lines of the NCI-9 melanoma cell lines.

Abstract LB-79: Synergistic anticancer effects of the 9.2.27PE immunotoxin and ABT-737 in melanoma involves silencing of the anti-apoptotic protein Mcl-1

Abstract In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE)-based immunotoxin, the 9.2.27PE, and the Bcl-2 family inhibitor ABT-737 in a panel of melanoma cell lines. ABT-737 inhibits the Bcl-2, Bcl-xl and the Bcl-w, but not Mcl-1 and A1 of the Bcl-2 family. The 9.2.27PE + ABT-737 drug combination resulted in synergistic cytotoxicity in the melanoma cells independent of BRAF mutation status. The cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. In addition, ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78, phosphorylated eIF2α protein levels and increased intracellular calcium levels. The increased intracellular calcium levels were enhanced by silencing of Mcl-1 by short hairpin RNA or by treatment with the 9.2.27PE immunotoxin. These results suggests that Mcl-1 has an inhibitory effect on calcium release from the ER, and that strongly decreased Mcl-1 protein levels after treatment with 9.2.27PE or shRNA may be responsible for the increased intracellular calcium levels in ABT-737 treated melanoma cells. Calcium release has been shown to take place through the inositol 1,4,5-triphosphate receptor in the ER membrane, and the Bcl-2 family proteins Bcl-2, Bcl-xl and Mcl-1 have been shown to interact with the inositol 1,4,5-triphosphate receptor, supporting our results.1 Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model. 1. Eckenrode EF et. Al., J Biol Chem. 2010 Apr 30;285(18):13678-84 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-79. doi:1538-7445.AM2012-LB-79

Abstract 2106: Long non-coding RNA signatures for melanoma detection in humans

Abstract Melanoma, an aggressive carcinoma that originates in the basal layer of human epidermis, has the highest mortality rate of skin cancers in industrialized countries. Unfortunately, current treatments for metastatic melanoma provide mostly palliative benefits to only a small proportion of patients. Despite the obvious need for improvement in assessment and treatment of metastatic melanoma, the intricate mechanisms underlying melanoma transformation remain unresolved and poorly understood. Thus, there is considerable interest in understanding the molecular basis of initiation and progression of human melanomas. Recent evidence indicates that long non-coding RNAs (lncRNAs), a subclass of noncoding RNA (ncRNA), are associated with carcinogenesis in melanoma as well as several other human cancers. The elucidation of the molecular function of these RNA species promises to bridge gaps in our knowledge of melanoma development in humans. The expression of selected lncRNAs (CR618740 and AF091731) in a panel of melanoma cell lines (pigmented and non-pigmented), melanocytes, and keratinocytes were assayed by DNA microarray (Ncode arrays from Lifetechnologies) and further confirmed by real-time polymerase chain reaction (qRT-PCR). Next, cell lines results were tested with patient samples. Our results indicate that the differential expression of lncRNAs is an indicator of the development of melanoma, and dysregulated expression of these markers may be involved in the transition of melanocytes to melanoma. Furthermore, the presence of significant correlation between melanoma cell line characteristics (stage, cell phenotype, types of mutations and others) and lncRNA expression data may imply novel causal connection between lncRNA expression and melanoma development. Importantly, this work will investigate the new possibility that lncRNAs play an essential role in cellular regulation and represent a potential platform for melanoma progression. Thus, unraveling the differential lncRNA expression in melanoma could initiate novel understanding of melanocyte homeostasis, tissue organization, and melanoma genesis in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2106. doi:1538-7445.AM2012-2106

Abstract LB-263: Synthetic lethal targeting of PTEN-associated homologous recombination (HR) deficient melanoma cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors

Abstract Background Mutation of the tumour suppressor PTEN is well-characterised in many advanced malignancies, including 30-50% of metastatic melanomas. Emerging evidence suggests a new role for PTEN in DNA repair, through transcriptional downregulation of the critical HR protein RAD51. Tumour cells deficient in HR (such as BRCA-deficient breast and ovarian cancers) have been therapeutically targeted in a synthetic lethality (SL) strategy using PARP inhibitors that block single strand break repair (SSBR), a pathway related to base excision repair (BER). We have previously identified novel inhibitors of the critical BER enzyme APE1, and demonstrated these can induce SL in BRCA-deficient cell lines. In the current study, we have evaluated SL relationships in PTEN-proficient and -deficient melanoma systems for a potential novel treatment strategy. Method We screened a panel of melanoma cell lines (MeWo, MM418, SkMel28, SkMel30, HT144, UACC62) for PTEN, APE1 and RAD51 protein expression. We tested for SL using clonogenic survival assays in PTEN- and RAD51-deficient cell lines using our recently developed APE1 DNA repair domain inhibitors, methoxyamine (indirect APE1 inhibitor) and NU1025 (PARP inhibitor), either alone or in combination. Aldehyde reactive probe assay, neutral comet assay, γH2AX immunofluorescence and FACS analyses were performed to confirm biological activity in cells. Results Of the screened cell lines, SkMel28, HT144 and UACC62 were deficient in PTEN and RAD51 protein expression. APE1 inhibitors were more toxic to PTEN-deficient cells compared to PTEN-proficient cells (p&amp;lt;0.01). Significant sensitivity was also seen in PTEN-deficient cells treated with methoxyamine (p&amp;lt;0.05) and PARP inhibitors (p&amp;lt;0.001), confirming that BER modulation results in SL in PTEN deficiency. This increased cytotoxicity in PTEN deficiency is associated with accumulation of DNA double strand breaks (DSB), as assessed by comet assay (p&amp;lt;0.001) and γH2AX accumulation (p&amp;lt;0.05). DSB accumulation leads to G2/M arrest and apoptosis, as demonstrated by FACS analysis. Conclusion Our data provides the first pre-clinical evidence that targeting APE1 in PTEN-RAD51 deficient melanoma cells is a promising new SL strategy. We are complementing the current small molecule studies by developing PTEN knockout systems using shRNA technology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-263. doi:1538-7445.AM2012-LB-263

Abstract 4893: XIAP downregulation accompanies inhibition of melanoma xenograft growth by mebendazole

Abstract Background: Melanoma's intrinsic resistance to therapy has been attributed to modulation of cellular survival pathways including apoptosis. We originally reported that mebendazole (MBZ), which acts as a microtubule-disrupting agent, is a potent inducer of apoptosis in melanoma cells in vitro via a Bcl-2 dependent mechanism (Molecular Cancer Research, 2008). Here, we show that MBZ is also able to effectively modulate another key anti-apoptotic factor, XIAP, to promote melanoma apoptosis both in vitro and in vivo. Methodology: Safety and efficacy of mebendazole was assessed using a human melanoma xenograft model established from M-14 cells. The effect of MBZ on XIAP expression and induction of apoptotic pathways was evaluated by immunoblotting and co-immunoprecipitation techniques along with densitometric quantification. The growth inhibitory effects of MBZ were determined in a panel of melanoma cell lines with the sulphorhodamine (SRB) assay. siRNA techniques were used to reduce expression of XIAP. Results: Oral MBZ is as effective as the current standard of care temozolomide (TMZ) in reducing melanoma growth in vivo, with no notable toxicities observed. Inhibition of melanoma growth in vivo is accompanied by MBZ specific reduced expression of XIAP and cleavage of caspase 9. In response to MBZ treatment, sensitive cell lines display reduced levels of XIAP expression whereas resistant cell lines do not. Moreover, down-regulation of XIAP expression by siRNA in the MBZ-resistant melanoma cell line UACC1097 was accompanied by a reduction in the IC50 for MBZ from &amp;gt;10 μM to 3.7 μM. Exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating inhibition of apoptosis by XIAP. Conclusions: In addition to demonstrating efficacy and safety in vivo, our preclinical studies of the promising anti-melanoma agent mebendazole provide insight into characterization of an apoptotic promoting mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4893. doi:1538-7445.AM2012-4893

Abstract 4170: Mutation and chemosensitivity profiling of 18 human melanoma cell lines

Abstract Malignant melanomas account for a substantial proportion of cancers worldwide and are largely resistant to conventional therapy. The heterogeneity and the invasiveness of melanomas present notable challenges for anti-cancer treatment. Recently, several mutations in RAF, RAS, and PTEN were identified that allow for prognosis of cancer development and progression. Therefore, inhibitors of the RAF-MEK-ERK pathway form attractive candidates for molecular-based targeted treatment of melanoma cancer. Here, we present the molecular characterization and chemosensitivity profiling of 18 human melanoma cell lines. Eight of these were established by Oncotest from human tumor xenografts originally derived from primary patient material. The molecular profiling included mutational analysis for BRAF, PIK3CA, KRAS, NRAS, TP53, and PTEN by sequencing. The chemosensitivity profiles were determined in vitro using a fluorescence-based cytotoxicity assay and included commonly used chemotherapeutic agents for treatment of melanoma, targeted agents (sorafenib and bortezomib) and various BRAF (PLX-4032, PLX-4720, GDC-0879) and MEK (CI-1040, PD0325901) inhibitors. The clinically relevant BRAF V600E substitution was the most prevalent mutation and was found in more than 50% of the melanoma cell lines. The chemosensitivity profiling revealed diverse patterns of selectivity and potency with mean IC50 values between 0.4 nM (vinblastine) and 8.3 µM (carboplatin). Each of the BRAF inhibitors tested was clearly more active in melanoma cell lines carrying a BRAF mutation. On average, IC50 values of BRAF inhibitors were more than 20-fold lower for the BRAF V600E mutated cell lines compared to the wild type cell lines. However, the BRAF mutated cell line MEXF 462NL was resistant towards BRAF inhibitors and it is warranted to compare its gene expression profile and mutational status with BRAF mutated cell lines responding to BRAF inhibitors. By using an ex vivo clonogenic assay, similar activity patterns for BRAF inhibitors were found, with mutated BRAF tumor xenografts showing markedly higher sensitivity. Potential synergistic activity of BRAF inhibitors with other inhibitors of the RAF-MEK-ERK pathway or with standard chemotherapeutic agents will be presented. The mutational status of selected cancer related genes, as well as responsiveness towards many cytotoxic and targeted agents was analyzed for 18 melanoma cell lines. This extensive genotypic and phenotypic characterization makes this melanoma cell line panel a valuable tool for in vitro profiling of novel compounds, in particular inhibitors of the RAF-MEK-ERK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4170. doi:1538-7445.AM2012-4170

FOXP3 is not mutated in human melanoma

<i>FOXP3</i> is not mutated in human melanoma

A potent Chk1 inhibitor is selectively cytotoxic in melanomas with high levels of replicative stress

There are few effective treatments for metastatic melanoma. Checkpoint kinase 1 (Chk1) inhibitors are being trialled for their efficacy in enhancing conventional chemotherapeutic agents, but their effectiveness as single agents is not known. We have examined the effectiveness of two novel Chk1 selective inhibitors, AR323 and AR678, in a panel of melanoma cell lines and normal cell types. We demonstrate that these drugs display single-agent activity, with IC50s in the low nanomolar range. The drugs produce cytotoxic effects in cell lines that are most sensitive to these drugs, whereas normal cells are only sensitive to these drugs at the higher concentrations where they have cytostatic activity. The cytotoxic effect is the consequence of inhibition of S-phase Chk1, which drives cells prematurely from late S phase into an aberrant mitosis and results in either failure of cytokinesis or cell death through an apoptotic mechanism. The sensitivity to the Chk1 inhibitors was correlated with the level of endogenous DNA damage indicating replicative stress. Chk1 inhibitors are viable single-agent therapies that target melanoma cells with high levels of endogenous DNA damage. This sensitivity suggests that Chk1 is a critical component of an adaptation to replicative stress in these cells. It also suggests that markers of DNA damage may be useful in identifying the melanomas and potentially other tumour types that are more likely to be sensitive to Chk1 inhibitors as single agents.

Comparative Metabolic Flux Profiling of Melanoma Cell Lines: BEYOND THE WARBURG EFFECT

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.

Abstract A65: Targeting the defective decatenation checkpoint in melanoma.

Abstract In Australia melanomas account for 10% of all reported cancers and 3% of cancer mortalities. Current treatments offer &amp;lt;25% response rate and &amp;lt;8 months improved survival. Cancers frequently demonstrate defects in the cell cycle, particularly the checkpoint mechanisms. Loss of these protective mechanisms may provide opportunities for selectively targeting cancer cells. One G2 phase checkpoint, the decatenation checkpoint, ensures that entangled sister chromatids can be segregated with high fidelity during cellular replication. Topoisomerase II is responsible for resolving these tangles. Using ICRF-193, an inhibitor of Topoisomerase II, a panel of melanoma cell lines were investigated for decatenation checkpoint function. This analysis revealed that 17/25 melanoma cell lines possessed a defective decatenation checkpoint, suggesting it to be a common defect. Treatment with ICRF-193 caused checkpoint defective cells to undergo an aberrant mitosis, resulting in accumulation of cells exhibiting abnormal nuclear morphologies, while checkpoint functional cells remained relatively unaffected. Comparative gene expression analysis revealed differential expression of cell-division and cell-death associated genes, suggesting adaptive changes compensating for checkpoint failure. A strong gene-expression signature for the checkpoint defect was also observed in this analysis and potential bio-markers are being investigated for use identifying this defect in tumors. A synthetic lethality siRNA screen of the kinome (∼700 genes) was carried out to identify genes/pathways crucial to the viability of cell lines with a defective decatentation checkpoint. This screen identified 10 potential hits, many involved in PI3Kinase signaling. We have validated this pathway as a selective target. This research has shown that the defective decatenation checkpoint is a viable target in melanoma. Using targets identified by the synthetic lethality screens and bio-markers to the defect in tumors, it should be possible to identify tumors most likely to be sensitive to drugs targeting the checkpoint defect, providing effective and selective therapeutics for the treatment of metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A65.

Cross-Platform Array Screening Identifies COL1A2, THBS1, TNFRSF10D and UCHL1 as Genes Frequently Silenced by Methylation in Melanoma

Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.

The Novel Gamma Secretase Inhibitor RO4929097 Reduces the Tumor Initiating Potential of Melanoma

Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma.