Patient-derived Melanoma Cell Lines Research Articles

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination invivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro.

Cancer cell phenotype largely depends on oxygen availability. The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue. Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2). A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines. Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner. Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells. Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner. In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

Plasticity of Drug-Naïve and Vemurafenib- or Trametinib-Resistant Melanoma Cells in Execution of Differentiation/Pigmentation Program.

Melanoma plasticity creates a plethora of opportunities for cancer cells to escape treatment. Thus, therapies must target all cancer cell subpopulations bearing the potential to contribute to disease. The role of the differentiation/pigmentation program in intrinsic and acquired drug resistance is largely uncharacterized. MITF level and expression of MITF-dependent pigmentation-related genes, MLANA, PMEL, TYR, and DCT, in drug-naïve and vemurafenib- or trametinib-treated patient-derived melanoma cell lines and their drug-resistant counterparts were analysed and referred to genomic alterations. Variability in execution of pigmentation/differentiation program was detected in patient-derived melanoma cell lines. Acute treatment with vemurafenib or trametinib enhanced expression of pigmentation-related genes in MITF-Mhigh melanoma cells, partially as the consequence of transcriptional reprograming. During development of resistance, changes in pigmentation program were not unidirectional, but also not universal as expression of different pigmentation-related genes was diversely affected. In selected resistant cell lines, differentiation/pigmentation was promoted and might be considered as one of drug-tolerant phenotypes. In other resistant lines, dedifferentiation was induced. Upon drug withdrawal (“drug holiday”), the dedifferentiation process in resistant cells either was enhanced but reversed by drug reexposure suggesting involvement of epigenetic mechanisms or was irreversible. The irreversible dedifferentiation might be connected with homozygous loss-of-function mutation in MC1R, as MC1RR151C +/+ variant was found exclusively in drug-naïve MITF-Mlow dedifferentiated cells and drug-resistant cells derived from MITFhigh/MC1RWT cells undergoing irreversible dedifferentiation. MC1RR151C +/+ variant might be further investigated as a parameter potentially impacting melanoma patient stratification and aiding in treatment decision.

Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance.

Background: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood. Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes. Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance. Methods: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs. The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions. Results: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs. Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced. TGF-β transported by sEVs contributed to the promotion of a suppressive phenotype of APCs. Conclusion: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.

Abstract 826: Global DNA methylation levels regulate PD-L1 expression in melanoma

Abstract The programmed death-ligand 1 (PD-L1) receptor is an important immune checkpoint and is often upregulated in cancer cells to allow immune evasion. In melanoma, the patients with PD-L1 expression and absense of tumour infiltrating lymphocytes (TILs) (i.e. “constitutive PD-L1 or PD-L1CON”) show worse response rates and prognosis than patients with PD-L1 expression and the presence of TILs (i.e. “inducible PD-L1 or PD-L1IND”). However, how PD-L1 expression is regulated in melanoma cells remains elusive. Understanding the mechanisms of how PD-L1 is regulated is important for predicting responses for anti-PD-L1 treatment and for developing new combinatorial therapies. We hypothesised that epigenetic state regulates constitutive and inducible PD-L1 expression in melanoma. To address the hypothesis, we have generated whole-genome scale DNA methylomes (using reduced representation bisulfite sequencing) and transcriptomes (RNA-Seq) for patient derived melanoma cell lines (in PD-L1IND and PD-L1CON groups). We discovered extensive global hypomethylation in the constitutive lines, particularly pronounced in intergenic repeat regions and gene bodies. A high proportion of hypomethylated regions exhibited dichotomous methylation patterns indicating a common regulatory mechanism between the PD-L1IND and PD-L1CON lines. RNA-Sequening data indicated that the hypomethylated state of the PD-L1CON cells was correlated with higher upregulation of the differentially expressed genes at a global-scale and the upregulated genes were associated with cancer hallmark properties. The upregulated genes exhibited signatures of viral mimicry and cytosolic sensing of dsRNA similar to what has been observed after DNA methyltransferase inhibitor (DNMTi) treatment in cancers. DNMTi treatment increased PD-L1 transcription in the PD-L1IND cell lines. PD-L1CON samples showed greater resistance to DNMTi compared to PD-L1 negative samples. This was observed at protein-coding genes, transposable elements and long non-coding RNAs. Moreover, genes involved in the innate immune pathway was strongly increased upon DNMTi mediated hypomethylation in the PD-L1IND cell lines (FDR-adjusted P-value = 0.02), however there was no expression change in the PD-L1 positive cells (Adjusted P-value = 1.0). Furthermore, we found that the IRF1 transcription factor, which plays a critical role in the innate immune pathway and also binds to the PD-L1 promoter, is significantly upregulated upon DNMTi in the PD-L1 negative cell lines (Adjusted P-value = 0.01) but not in the PD-L1 positive cells (Adjusted P-value = 0.9). These results show that global hypomethylation levels regulate PD-L1 expression in melanoma. We believe these results are the first to show that DNA methylation levels play a role in regulating PD-L1 on melanoma and suggest they may have important implications for combined treatments targeting methylation (DNMTi) and PD1/PD-L1 (anti-PD1 antibodies). Citation Format: Aniruddha Chatterjee, Antonio Ahn, Euan J. Rodger, Peter A. Stockwell, Matthew Parry, Jyoti Motwani, Stuart J. Gallagher, Elena Shklovskaya, Jessamy Tiffen, Peter Hersey, Michael R. Eccles. Global DNA methylation levels regulate PD-L1 expression in melanoma [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 826.

Abstract 3946: Tumor checkpoint inhibitor profiling for an optimal clinical response

Abstract The last three decades have seen a steady rise in melanoma, the most aggressive form of skin cancer. The discovery of checkpoint inhibitors has revolutionized the treatment landscape for melanoma; anti-CTLA4 and anti-PD1 showing great successes in clinic. While checkpoint molecules and co-stimulatory molecules are traditionally present on cells involved in immune activation, mounting evidence suggests that tumor cells also express these molecules. Our laboratory has characterized five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 and screened them for the expression of a comprehensive list of 29 co-stimulatory and co-inhibitory molecules compared to normal adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. These findings were additionally validated in melanoma tissues, obtained from patients, by immunohistochemistry. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. This upregulation was coupled with an increase in transcription factor MITF, a binding site for which is present in the promoter region of all the upregulated molecules. Co-culture experiments with immune cells, demonstrate a modulatory role played by the HVEM/BTLA/CD160 axis molecules that are present on tumor cells. The presence of these additional immune-regulatory molecules signify the existence of a robust tolerance mechanism induced by these checkpoint inhibitors. Our studies underscore the importance of characterizing tumor profiles to enable the selection of an optimal treatment regimen combining targeted small molecule inhibitors, and checkpoint inhibitors to maximize T cell killing, and tumor clearance. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, John J. Degliuomini, Marc Wallack, Jan Geliebter, Raj K. Tiwari. Tumor checkpoint inhibitor profiling for an optimal clinical response [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 3946.

Somatic Hypermutation of the YAP Oncogene in a Human Cutaneous Melanoma.

Melanoma is usually driven by mutations in BRAF or NRAS, which trigger hyperactivation of MAPK signaling. However, MAPK-targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that co-operatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although is thought to be only rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of TAZ activity and also of classical melanoma driver mutations such as BRAF and NRAS. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four serine residues known to be targeted by the Hippo pathway and we show that they lead to hyperactivation of YAP. IMPLICATIONS: Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers additional to YAP protein expression.

Abstract B174: Co-expression of stimulators and inhibitors of T-cell activation in melanoma

Abstract Melanoma, one of the most aggressive skin cancers, has steadily been on the rise over the last three decades. With limited treatment options, the recent impetus in immuno-oncology drugs, especially checkpoint inhibitors, has changed the treatment landscape. While anti-CTLA4 and anti-PD1 demonstrated success in clinic, the search for additional targets is needed to enable potent comprehensive curative combination therapies. Our laboratory has characterized and screened five primary patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2. While immunomodulatory molecules are canonically present on antigen presenting cells and T-cells, increasing evidence suggests that they are not expressed in isolation. In an effort to identify immunomodulatory molecules expressed on our primary melanoma cells, a comprehensive RT-PCR screen of 29 co-inhibitory and co-stimulatory molecules was carried out. Several molecules including CD160, CD226, TIM1, HVEM, and BTLA were seen to be differentially expressed in melanoma cells compared to normal melanocytes at the mRNA level. Western blots and immunocytochemistry validated the differential expression of these molecules at the protein level. 50-80% of melanoma cases are positive for the BRAFV600E mutation, and are treated with a small molecule inhibitor of the mutated BRAF, vemurafenib (PLX4032). Treatment of these cells with PLX4032 led to an upregulation of transcription factors MITF and AP-1, as well as immunomodulatory molecules, CD160, CD226, TIM1, HVEM, and BTLA, a phenomenon seen only in cells positive for the BRAFV600E lesion. MITF and AP-1, owing to the binding sites present in the promoter regions of these molecules, can drive their expression upon treatment with PLX4032. These additional immune-regulatory molecules of T-cell activation and/or immune tolerance mechanisms are potential targets for a combination therapy with PLX4032 in melanoma patients positive for the BRAFV600E genetic lesion. Our future directions aim to elucidate the role of these molecules on the tumor cells and devise an effective combination with small-molecule inhibitors and immunotherapies. Citation Format: Rachana Maniyar, Robert Freund, Aryan Malhotra, Sanjukta Chakraborty, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Co-expression of stimulators and inhibitors of T-cell activation in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B174.

Brain Distribution and Active Efflux of Three panRAF Inhibitors: Considerations in the Treatment of Melanoma Brain Metastases.

Targeted inhibition of RAF and MEK by molecularly targeted agents has been employed as a strategy to block aberrant mitogen-activated protein kinase (MAPK) signaling in melanoma. While the use of BRAF and MEK inhibitors, either as a single agent or in combination, improved efficacy in BRAF-mutant melanoma, initial responses are often followed by relapse due to acquired resistance. Moreover, some BRAF inhibitors are associated with paradoxical activation of the MAPK pathway, causing the development of secondary malignancies. The use of panRAF inhibitors, i.e., those that target all isoforms of RAF, may overcome paradoxical activation and resistance. The purpose of this study was to perform a quantitative assessment and evaluation of the influence of efflux mechanisms at the blood-brain barrier (BBB), in particular, Abcb1/P-glycoprotein (P-gp) and Abcg2/breast cancer resistance protein (Bcrp), on the brain distribution of three panRAF inhibitors: CCT196969 [1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea], LY3009120 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea, and MLN2480 [4-pyrimidinecarboxamide, 6-amino-5-chloro-N-[(1R)-1-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-]. In vitro studies using transfected Madin-Darby canine kidney II cells indicate that only LY3009120 and MLN2480 are substrates of Bcrp, and none of the three inhibitors are substrates of P-gp. The three panRAF inhibitors show high nonspecific binding in brain and plasma. In vivo studies in mice show that the brain distribution of CCT196969, LY3009120, and MLN2480 is limited, and is enhanced in transgenic mice lacking P-gp and Bcrp. While MLN2480 has a higher brain distribution, LY3009120 exhibits superior in vitro efficacy in patient-derived melanoma cell lines. The delivery of a drug to the site of action residing behind a functionally intact BBB, along with drug potency against the target, collectively play a critical role in determining in vivo efficacy outcomes.

Abstract 714: A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells

Abstract Metastatic melanoma, leads to the highest number of skin cancer related deaths. Checkpoint inhibitor therapy has witnessed a high success rate in melanoma patients with anti-CTLA-4 and anti-PD-1. However, checkpoint inhibitor molecules and their compensatory stimulatory counterparts are widely expressed on T cells and antigen presenting cells suggesting a robust redundancy in these molecules as clinical targets. Some of these molecules that include CTLA-4, PD-1, HVEM, VISTA, 41-BB, OX-40 and CD226 are also expressed on tumor cells. Their role in regulating an immune response whether it is cell killing or immune evasion remains to be elucidated. We isolated and characterized five primary patient derived melanoma cell lines: MEL-2, MEL-V, 3MM, KFM and GLM2. We screened these cells for the expression of a comprehensive panel of twenty-five co-stimulatory and co-inhibitory molecules by RT-PCR which revealed significant heterogeneity in expression of these molecules compared to normal adult melanocytes under normal conditions; underscoring the importance of understanding tumor tissue pleiotropy prior to designing a therapeutic regimen. Surprisingly, inhibitory molecules including PD-1, VISTA and LAIR1 and stimulatory molecules including 4-1BB, HVEM and ICOS were upregulated differentially in these cell lines by metabolic stress brought on by starvation conditions. Some of these molecules were restored to basal levels of expression on treatment with 10μM vemurafenib (PLX4032), a BRAFV600E inhibitor, for 24 hours. However the treatment led to concurrent upregulation of molecules such as LAG3, BTLA, CD226 and TIM1, suggesting a compensatory mechanism that could aid melanoma adaptation and escape from immune recognition. Exposing melanoma cells to classical activated dendritic cell cytokines, IL-6 and IL-12, led to a differential expression of these molecules. Additionally, experiments using tumor lysate loaded dendritic cells to study activation revealed an ability to modulate immune activation correlating with unique stimulatory and inhibitory molecule expression profile of each primary cell line. Our results underscore the importance of understanding the profile of co-stimulatory and co-inhibitory molecules expressed in tumor cells. With eighty percent of melanoma patients being positive for the BRAFV600E lesion, we make a case for designing a combinatorial therapeutic regimen, with targeted immunotherapies as well as targeting specific genetic lesions with small molecule inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Marc Wallack, Jan Geliebter, Raj K. Tiwari. A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 714.

The Effect of Topoisomerase I Inhibitors on the Efficacy of T-Cell-Based Cancer Immunotherapy.

BackgroundImmunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy.MethodsTwo pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided.ResultsWe found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett’s test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor–treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey’s test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test).ConclusionsWe demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.

The unfolded protein response impacts melanoma progression by enhancing FGF expression and can be antagonized by a chemical chaperone

The mechanisms hallmarking melanoma progression are insufficiently understood. Here we studied the impact of the unfolded protein response (UPR) - a signalling cascade playing ambiguous roles in carcinogenesis - in melanoma malignancy. We identified isogenic patient-derived melanoma cell lines harboring BRAFV600E-mutations as a model system to study the role of intrinsic UPR in melanoma progression. We show that the activity of the three effector pathways of the UPR (ATF6, PERK and IRE1) was increased in metastatic compared to non-metastatic cells. Increased UPR-activity was associated with increased flexibility to cope with ER stress. The activity of the ATF6- and the PERK-, but not the IRE-pathway, correlated with poor survival in melanoma patients. Using whole-genome expression analysis, we show that the UPR is an inducer of FGF1 and FGF2 expression and cell migration. Antagonization of the UPR using the chemical chaperone 4-phenylbutyric acid (4-PBA) reduced FGF expression and inhibited cell migration and viability. Consistently, FGF expression positively correlated with the activity of ATF6 and PERK in human melanomas. We conclude that chronic UPR stimulates the FGF/FGF-receptor signalling axis and promotes melanoma progression. Hence, the development of potent chemical chaperones to antagonize the UPR might be a therapeutic approach to target melanoma.

Abstract 3656: Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells

Abstract Melanoma is one of the deadliest forms of skin cancer, with a dim prognosis when metastasized, leading to the highest number of skin cancer related deaths. In recent years there has been a focus on the use of checkpoint inhibitor therapies like anti-CTLA-4, anti-PD-1 and anti-PD-L1 to treat melanoma. PD-L1, HVEM and VISTA expression on cancer cells has been shown to promote immune evasion and tumor survival. CTLA-4, when engaged on tumor cells leads to their apoptosis and LIGHT signaling leads to recruitment of T cells and effective tumor clearance. Thus, since these molecules do not function in isolation, we need to consider tissue pleiotropy and the expression of co-stimulatory and co-inhibitory molecules on tumor tissue. Our laboratory has characterized and established five primary patient derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM and GLM-2. In an effort to understand tumor tissue pleiotropy, we conducted a comprehensive expression pattern screening of eight co-inhibitory and ten co-stimulatory molecules by RT-PCR. Among others, VISTA, a CD4+ T cell suppressor, HVEM, an immune evasion regulator, LAG3 and TIM3, potential immune checkpoint targets, were seen to be differentially expressed in these primary cell lines. Current therapies target CTLA-4, PD-1 and PD-L1, which also exhibit varying expression in our primary cell lines, pointing to the importance of considering tissue expression of these molecules when administering these novel immunotherapies. Additionally, 80% of melanoma patients are positive for the BRAFV600E genetic lesion and are administered vemurafenib (PLX4032), an inhibitor of the overactive mutated BRAF. Our in vitro studies show that treatment with PLX4032, changed the expression of CTLA-4 and PD-L1 in these primary cells. We observed a decrease in PD-L1 protein expression in MEL-V and GLM-2 and an increase in PD-L1 protein expression in MEL-2 and KFM, the four BRAFV600E positive cell lines. CTLA-4 protein expression demonstrated an upward trend on treatment with PLX4032. This synergy observed between treatment with drugs targeting genetic lesions and the expression of immunomodulatory molecules warrants characterization of tumor biopsies prior to designing an effective combinatorial therapy regime. Our long term goal is to optimize combinatorial immune and drug therapies directed against both co-stimulators and checkpoint inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Tuli, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells [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 3656. doi:10.1158/1538-7445.AM2017-3656

Abstract 2069: Overcoming drug resistance by targeting melanoma dedifferentiation through information-theoretic analysis and single cell proteomics

Abstract Despite impressive initial response of BRAF targeted therapy in mestastic melanoma patients, acquired drug resistance, often times derived from tumor heterogeneity, always limit its clinical outcome. A dedifferentiation process named melanocyte to neural crest transition (MNT) has shown to be critical in the early stage resistance development. The dedifferentiation could induce a whole cell state change resulting a drug tolerant state of melanoma cells, which will lead to initial drug resistance. Mathematic modeling indicates the nature of the transition as results of both stochastic phenotypic transition and non-genetic drug selection, which indicates and further proved that the whole cell state transition should be reversible during drug removal. Information theoretic analysis reduced the dimensional of thousands of gene expression level changes as results of only two unbalanced processes onto a cellular steady state. Reversible transition trajectory defined through two processes indicates it to resemble a critical transition in physical systems. Refined single cell proteomic analysis of those proteins that associate with major unbalanced processes identify the critical points of the transition as early as day6, which cannot be resolved from bulk proteomic measurement. By co-targeting the drivers at the critical points in combination with original brafi targeted drug successfully arrested the transition and induced a sustained tumor growth inhibition in multiple patient-derived melanoma cell lines. This study provides us a noval methodology of mining whole transcriptomic transition with information theoretic analysis to reduce the dimensionality and then using single cell proteomic analysis for refining critical points and driver proteins which eventually rationalized a effective combination therapy to stop the drug resistance development early on. Citation Format: Yapeng SU, Wei Wei, Lidia Robert, Jennifer Tsoi, Min Xue, Jungwoo Kim, Thomas Graeber, Raphael Levine, Antoni Ribas, James Heath. Overcoming drug resistance by targeting melanoma dedifferentiation through information-theoretic analysis and single cell proteomics [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 2069. doi:10.1158/1538-7445.AM2017-2069

Multiple Isoforms of ANRIL in Melanoma Cells: Structural Complexity Suggests Variations in Processing.

The long non-coding RNA ANRIL, antisense to the CDKN2B locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms. Despite the identification of multiple isoforms of ANRIL, expression of certain transcripts has been found to be tissue-specific and the characterisation of ANRIL transcripts remains incomplete. Several functions have been associated with ANRIL. In our judgement, studies on ANRIL functionality are premature pending a more complete appreciation of the profusion of isoforms. We found differential expression of ANRIL exons, which indicates that multiple isoforms exist in melanoma cells. In addition to linear isoforms, we identified circular forms of ANRIL (circANRIL). Further characterisation of circANRIL in two patient-derived metastatic melanoma cell lines (NZM7 and NZM37) revealed the existence of a rich assortment of circular isoforms. Moreover, in the two melanoma cell lines investigated, the complements of circANRIL isoforms were almost completely different. Novel exons were also discovered. We also found the family of linear ANRIL was enriched in the nucleus, whilst the circular isoforms were enriched in the cytoplasm and they differed markedly in stability. With respect to the variable processing of circANRIL species, bioinformatic analysis indicated that intronic Arthrobacter luteus (Alu) restriction endonuclease inverted repeats and exon skipping were not involved in selection of back-spliced exon junctions. Based on our findings, we hypothesise that “ANRIL” has wholly distinct dual sets of functions in melanoma. This reveals the dynamic nature of the locus and constitutes a basis for investigating the functions of ANRIL in melanoma.

P90RSK Blockade Inhibits Dual BRAF and MEK Inhibitor-Resistant Melanoma by Targeting Protein Synthesis

Despite improvements in survival in metastatic melanoma with combined BRAF and mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor treatment, the overwhelming majority of patients eventually acquire resistance to both agents. Consequently, new targets for therapy in resistant tumors are currently being evaluated. Previous studies have identified p90 subfamily of ribosomal S6 kinase (p90RSK) family kinases as key factors for growth and proliferation, as well as protein synthesis via assembly of the 7-methyl-guanosine triphosphate cap-dependent translation complex. We sought to evaluate inhibitors of p90RSK family members: BI-D1870 and BRD7389, for their ability to inhibit both proliferation and protein synthesis in patient-derived melanoma cell lines with acquired resistance to combined treatment with the BRAF inhibitor vemurafenib and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor selumetinib. We found that the RSK inhibitors blocked cell proliferation and protein synthesis in multiple dual-resistant melanoma lines. In addition, single agent RSK inhibitor treatment was effective in drug-naïve lines, two of which are innately vemurafenib resistant. We also used Reverse Phase Protein Array screening to identify differential protein expression that correlates with BI-D1870 sensitivity, and identified prognostic biomarkers for survival in human melanoma patients. These findings establish p90RSK inhibition as a therapeutic strategy in treatment-resistant melanoma and provide insight into the mechanism of action.

Autophagy inhibitors chloroquine and LY294002 enhance temozolomide cytotoxicity on cutaneous melanoma cell lines in vitro.

Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.

The role of autophagy inhibition in the enhanced cytotoxicity of temozolomide on melanoma cell lines

Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ) and LY-294.002 (LY) in temozolomide (TMZ) cytotoxicity in human melanoma cell lines. Materials and methods . The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated. Results . CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity. Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.

Abstract 2365: Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy

Abstract Over 1 million cases of skin cancer are diagnosed in the US every year, of which 75% deaths result due to melanoma, one of the deadliest forms of skin cancer, arising from melanocytes. We have isolated five patient derived melanoma cell lines (MEL-V, 3MM, GLM2, Mel2, KFM) and characterized the expression of various melanoma associated antigens (MAAs). The presence of a multitude repertoire of common MAAs (Gp100, MART-1, MAGE-A1, NY-ESO-1, Tyrosinase, TRP-1 (Gp75), TRP-2, Melanotransferrin, CD71, CD146) enabled us to use composite cell membrane preparations of these cells as a multivalent vaccine. To this end, we designed a vaccinia virus vaccine that was composed of membrane lysate preparations of the five primary cells and recombinant IL-2. When tested in a pilot study, delayed type hypersensitivity and a multivalent immune response were noted. In an effort to correlate genetic lesions with MAA expression we observed that in BRAF mutant melanoma cells, treatment with a BRAFv600E inhibitor, PLX4032, enhanced the expression of MAAs making the melanoma cells more visible to the vaccination driven immune response. We also observed that melanoma cells constitutively expressed CTLA-4 as determined by Immunofluorescence and Western Blots. This expression was found on the membranes as well as in the cytoplasm in comparatively high levels on wild type as well as the BRAFV600E mutant melanoma cell lines. Further our results suggest that CTLA-4 expression on melanoma cells promotes the immunosuppressive tumor microenvironment enabling tumor evasion. Given that CTLA-4 monoclonal antibodies such as Ipilimumab are approved for clinical treatment, this study opens up a new avenue into a plausible combinatorial therapy for our vaccinia virus based vaccine with Ipilimumab for BRAF mutant as well as wild type melanoma cells. Citation Format: Rachana R. Maniyar, Neha Tuli, Robert Suriano, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy. [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 2365.

Abstract 879: Single cell analysis resolves combinatory targeted therapy for arresting the BRAFi-induced cellular dedifferentiation of metastatic melanomas

Abstract Targeted therapy that inhibits the BRAF (V600E) protein has shown impressive initial responses in metastatic melanoma patients. However, acquired drug resistance, derived from tumor heterogeneity, always limits its efficacy and compromises its treatment outcomes. Melanocyte to neural crest transition (MNT) has been observed to play crucial role in early stage adaptive drug tolerance, and the mathematic modeling indicates the transition is contributed from both stochastic phenotypic transition and non-genetic selection. Whole transcriptomic analysis enriched important signaling proteins that are involved in the resistance development process. Single cell proteomic analysis of those proteins was performed on patient-derived melanoma models to further study the early stage drug tolerance. This resolves how protein signaling network coordination rewires in the process, which cannot be resolved from proteomic analysis of bulk tumor tissue. Signaling network coordination analysis indicates that co-inhibiting the hubs of the network (MEK, NFKB-p65) could keep cells in the drug-susceptible stages. The combination therapy that co-inhibits mutant BRAF, MEK and NFKB-p65 together successfully arrested the MNT transition and induced a sustained tumor growth suppression in multiple patient-derived melanoma cell lines. This study provides us a brand-new way of using single cell proteomic analysis to infer effective combinations by resolving protein signaling network coordination that is inaccessible through traditional bulk-level proteomic measurement. Citation Format: Yapeng Su, Wei Wei, Min Xue, Lidia Robert, Jennifer Tsoi, Thomas Graeber, Antoni Ribas, James Heath. Single cell analysis resolves combinatory targeted therapy for arresting the BRAFi-induced cellular dedifferentiation of metastatic melanomas. [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 879.