Parental Melanoma Cell Line Research Articles

Abstract LB019: Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity

Abstract Emerging sets of single-cell sequencing data makes it appealing to apply existing tumor phylogeny reconstruction methods to analyze associated intratumor heterogeneity. Unfortunately, tumor phylogeny inference is an NP-hard problem and existing principled methods typically fail to scale up to handle thousands of cells and mutations observed in emerging single-cell data sets. Even though there are greedy heuristics to build hierarchical clustering of cells and mutations, they suffer from well-documented issues in accuracy. Additionally even when “optimal” solutions are feasible, existing approaches only provide a single “most likely” tree to depict the evolutionary processes that may result in an observed collection of cells and mutations. To make matters worse, the vast majority of single-cell sequencing data sets are transcriptomic and as a result, suffer from considerable variation in coverage across mutational loci. In this paper, we introduce Trisicell, a computational toolkit for scalable tumor phylogeny reconstruction and validation from single-cell genomic, exomic or transcriptomic sequencing data. Trisicell has three components: (i) Trisicell-DnC, a new tumor phylogeny reconstruction method from genotype matrices derived from single-cell data, (ii) Trisicell-ConT a new algorithm for constructing the consensus for two or more tumor phylogenies - which may be built through the use of different data types on the same set of cells, or built through the use of different methods on the same data, and (iii) Trisicell-PF, a new partition function method for assessing the likelihood of any user-defined subtree/set of cells to be seeded by a given set of mutations in the phylogeny. Collectively, these tools provide means of identifying and validating robust portions of a tumor phylogeny, offering the ability to focus on the most important (sub)clones and the genomic alterations that seed the associated clonal expansion. We applied Trisicell to a panel of clonal sublines derived from single-cells of a parental mouse melanoma model on which we performed both whole exome and whole transcriptome sequencing. The tumor phylogenies of the clonal sublines built on exomic and transcriptomic mutations by Trisicell-DnC, were shown by Trisicell-ConT to be highly similar and the subtrees comprised of phenotypically similar clonal sublines were shown to be strongly associated by Trisicell-PF to their seeding mutations. In addition, we applied Trisicell to single-cell whole transcriptome sequencing data from a tumor derived from the same parental melanoma cell line, which was subjected to anti-CTLA-4 immunotherapy. The phylogenies generated from both studies featured distinct subtrees, strongly associated with phenotypes including cell differentiation status, tumor growth and therapeutic response. These results suggest that Trisicell can be used for scalable tumor phylogeny reconstruction and validation through both single-cell and clonal-subline sequencing data, which may reveal strong phenotypic associations. In particular, they suggest that the developmental status and phenotypic intratumoral heterogeneity of melanoma originates from observable subclonal variation. Citation Format: Farid Rashidi Mehrabadi, Salem Malikić, Kerrie L. Marie, Eva Pérez-Guijarro, Erfan Sadeqi Azer, Howard H. Yang, Can Kızılkale, Charli Gruen, Huaitian Liu, Christina Marcelus, Aydın Buluç, Funda Ergün, Maxwell P. Lee, Glenn Merlino, Chi-Ping Day, S. Cenk Sahinalp. Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB019.

Studies on imidazo[2,1-b][1,3]benzothiazole derivatives as new radiosensitizers

The synthesis and characterization of potent 7-substituted-2-(4-substitutedphenyl)imidazo[2,1-b][1,3]benzothiazoles as effective radiosensitizers and anticarcinogenic compound is reported. The activity is determined against human liver cancer Hep G2 cell line, two parental melanoma cell lines (human melanoma cell line, A375C and mouse melanoma cell line, A375C). The compounds 7-sulfonamide-2-(4-fluorophenyl)imidazo[2,1-b][1,3]benzothiazole (3f, IC50 = 0.097 μM) and 7-sulfonamide-2-(4-methylphenyl)imidazo[2,1-b][1,3]benzothiazole (3g, IC50 = 0.114 μM) exhibited considerable in vitro anticancer activity against Hep G2 cell line while 7-bromo-2-(4-fluorophenyl)imidazo[2,1-b][1,3]benzothiazole showed effectiveness against both parental melanoma cell lines (3c, IC50 = 0.04 and 0.052 μM). Further the active molecules 3f, 3g and 3h are tested for radiosensitizing activity over Hep G2 cell line at 4 Gy, with the comet formed at 0.054 μM . Similarly, 3c tested against two parental melanoma cell lines, it has proven to be more potent when combined with 2 Gy ϒ-radiation. The present study reveals that presence of sulfonamide in combination with methoxy substitution enhanced DNA fragmentation and there by acting as effective derivative for Hepatocellular carcinoma.

Abstract 1180: Quantifying melanoma drug resistance and tumor heterogeneity by live cell interferometry

Abstract Melanoma is the most aggressive type of skin cancer. Tumor heterogeneity and drug resistance are significant obstacles to survival benefits from mutation-targeted therapy. Over 50% of metastatic melanomas harbor V600BRAF mutations. Targeting of MUTBRAF melanoma with BRAF inhibitor monotherapy or the combination of BRAF and MEK inhibitors leads to a high rate of initial responses, but adaptive and acquired resistance frequently leads to clinical relapse. Although the presence of V600BRAF mutations guides the selection of therapy with MAPK inhibitors, methods to track preexisting and adaptive resistance and thereby predict response patterns, or to use this new information to modify therapy, are still lacking. Here, we aimed to develop a rapid, massively parallel tumor cell profiling method, based on Live Cell Interferometry (LCI), to quantify heterogeneous single tumor cell responses and emergent drug resistance. LCI is an ex vivo imaging approach that quantifies changes in total cell biomass, biomass motion, or cell stiffness over time. We report a novel high-throughput screening version of the LCI platform, HSLCI, which rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive, parental melanoma cell lines and their isogenic, BRAFi-resistant sub-lines. We show reproducible results from two different HSLCI platforms at two institutions and generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 hours. Our measurements require no fluorescence or dye labeling and are faster than field-standard growth inhibition assays. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections. Citation Format: Kevin A. Leslie, Dian Huang, Graeme Murray, Daniel Guest, Irena J. Roy, Marco Piva, Gatien Moriceau, Roger S. Lo, Michael A. Teitell, Jason C. Reed. Quantifying melanoma drug resistance and tumor heterogeneity by live cell interferometry [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 1180.

Abstract 4900: Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma

Abstract We aim to identify whether reactive oxygen species (ROS) targeted therapy using a ROS scavenger, A100, will inhibit tumor growth and evade BRAF inhibitor induced resistance in melanoma cells harboring the endogenous BRAFV600E mutation. We also propose to investigate the mechanism by which A100 sensitizes the dabrafenib-mediated resistance. Dabrafenib-resistant (DR) melanoma cells (WM-115DR, SK-MEL-24DR and A375DR) were generated using increasing doses of dabrafenib. We conducted flow cytometry experiments to measure ROS levels before and after acquisition of resistance. DCF-DA assay indicated upregulation of hydrogen peroxide levels in dabrafenib resistant cell lines versus parental cell lines. Furthermore, dabrafenib resistant cell lines demonstrated elevated superoxide levels as analyzed by MitoSOX. Crystal violet and three-dimensional matrigel assays were performed to analyze the effect of A100 and dabrafenib on cell growth of dabrafenib resistant and parental melanoma cell lines. We observed a statistically significant decrease in cell proliferation and colony formation of dabrafenib resistant cells when subjected to the combination of A100 and dabrafenib compared to single agent dabrafenib or A100. We also noted a trend of inhibition of cell proliferation in response to combined treatment of A100 and dabrafenib in parental cell lines versus the single agent. There was significant reduction in colony formation under similar condition in parental cells. Dabrafenib as a single agent had similar effect in reducing phosphorylated ERK1/2 as compared to co-treatment with A100 in A375, SK-MEL-24 and WM-115 parental cells. As expected, there was no alteration of MAPK signaling in resistant WM-115DR, SK-MEL-24DR and A375DR cells upon treatment with dabrafenib. A100 has been reported to cause DNA damage in leukemia cells. We analyzed the effect of A100 in the presence or absence of dabrafenib on markers of DNA damage. The data suggested that DNA damage is induced by A100 as assessed by increased levels of p-ATM, p-ATR and γ-H2AX. Further, A100 in combination with dabrafenib increased levels of p-ATM, p-ATR and γ-H2AX in parental and dabrafenib resistant cell lines. Proteomics analysis on WM-115 versus WM-115DR cell line was performed to determine alterations in mitochondrial protein expression upon resistance to BRAF inhibitors. The data indicated upregulation of proteins with antioxidant functions including SOD2 (superoxide dismutase [Mn]) and Peroxiredoxin-1 (PRDX1) in WM-115DR cells compared to WM-115 parental which was confirmed by immunoblotting analysis. Overall, this study suggested that the combination of ROS quenching agent (A100) with BRAF inhibitor (dabrafenib) could be a potential strategy to treat BRAF-mutant melanoma patients. Citation Format: Long Yuan, Rosalin Mishra, Hima Patel, Samar Alanazi, Joan Garrett. Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma [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 4900.

Abstract 2451: Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27

Abstract Evading apoptosis and limitless replicative potential are two hallmarks of malignant cells. Here we report that melanoma cells surviving the induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77 have even higher proliferative potential, and that this is associated with the increased expression of the long noncoding RNA (lncRNA) OVAAL which competes with p27 mRNA for binding to polypyrimidine tract binding protein 1 (PTBP1) and thus represses p27 mRNA translation. Prolonged exposure to TRAIL or Mcl-1 resulted in stable melanoma cell populations with increased proliferation activity. Comparative RNA-seq analysis identified OVAAL as the most significantly upregulated lncRNA in melanoma cells resistance to TRAIL and Mcl-1. ShRNA knockdown of OVVAL reversed the increased proliferative potential of resistant cells, and also inhibited proliferation in a subset of parental melanoma cell lines that constitutively expressed relatively high levels of OVAAL. RNA pulldown followed by Mass Spectrometry analysis revealed that among proteins that were physically associated with OVAAL was a protein called PTBP1 that bound to and enhanced the activity of the internal ribosomal entry site (IRES) in the 5' untranslated region (5'-UTR) of p27 mRNA. Indeed, OVAAL knockdown resulted in an increase in the association between PTBP1 and p27 IRES leading to upregulation of the p27 protein, whereas overexpression of OVAAL caused reduction in binding of PTBP1 to the IRES of p27. Furthermore, co-knockdown of PTBP1 abolished upregulation of p27 triggered by OVAAL knockdown. These results suggest that OVAAL competes with p27 mRNA for binding to PTBP1 and thus inhibits p27 expression and promotes cell proliferation. This was substantiated by binding assays using in vitro-synthesized biotin-labelled OVAAL, p27 IRES and purified PTBP1 in a cell-free system. Our results showed that the increased c-Myc activity was responsible for upregulation of OVAAL in melanoma cells selected for resistance to apoptosis induced by TRAIL or UMI-77. Collectively, these results identify the expression of OVAAL as an important mechanism promoting melanoma cell proliferation, with potential implications for targeting OVAAL in combination with apoptosis-inducing therapeutics in the treatment of melanoma. Citation Format: YUNA YUAN ZHANG, Xu Guang Yan, Margaret Farrelly, Hamed Yari, Yuchen Feng, Ting La, Hessam Tabatabaee, Lei Jin, Xu Dong Zhang. Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27 [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 2451.

High-Speed Live-Cell Interferometry: A New Method for Quantifying Tumor Drug Resistance and Heterogeneity.

We report the development of high-speed live-cell interferometry (HSLCI), a new multisample, multidrug testing platform for directly measuring tumor therapy response via real-time optical cell biomass measurements. As a proof of concept, we show that HSLCI rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive parental melanoma cell lines and in their isogenic BRAFi-resistant sublines. We show reproducible results from two different HSLCI platforms at two institutions that generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 h. Like other quantitative phase imaging (QPI) modalities, HSLCI is well-suited to noninvasive measurements of single cells and cell clusters, requiring no fluorescence or dye labeling. HSLCI is substantially faster and more sensitive than field-standard growth inhibition assays, and in terms of the number of cells measured simultaneously, the number of drugs tested in parallel, and temporal measurement range, it exceeds the state of the art by more than 10-fold. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.

The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3′-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.

GnT-V expression and metastatic phenotypes in macrophage–melanoma fusion hybrids is down-regulated by 5-Aza-dC: Evidence for methylation sensitive, extragenic regulation of GnT-V transcription

β1,6-acetylgulcosaminyltransferase V (GnT-V) forms β1,6 branching on the trimannosyl terminus of N-glycans, allowing for the production of β1,6Glc-NAc-bearing oligosaccharides. These are used by healthy myeloid cells and cancer cells alike for systemic migration. GnT-V has multiple glycoprotein substrates and thereby exerts global effects on cancer progression, characteristic of a master regulator of metastasis. Yet little is known of the regulation of GnT-V expression by tumor cells. It was previously reported that fusion of macrophages with Cloudman S91 mouse melanoma cells produced macrophage–melanoma hybrids with up-regulated GnT-V expression regarding mRNA and enzymatic activity. Majority of these hybrids showed increased chemotactic motility in vitro and elevated metastatic potential in vivo. Here we attempted to understand this at the molecular genetic level focusing on DNA hypermethylation as a potentially key step. Treatment of cells with 5-Aza-dC, an inhibitor of DNA methylation, resulted in decreased expression of GnT-V mRNA and β1,6-branched oligosaccharides along with reduced glycosylation of LAMP-1, a major substrate for GnT-V. This was accompanied by reduced chemotactic motility of the cells. The results suggested that DNA hypermethylation in some fashion stimulated GnT-V expression. We thus investigated the promoter region of the GnT-V gene for hypermethylation of CpG islands, comparing macrophage-melanoma hybrids of low and high metastatic potential with the parental melanoma cell line. Genomic DNA after bisulfite modification amplified from this region showed identical sequences between the cell lines. The findings indicated that differential methylation of the promoter region of GnT-V gene was not responsible for its transcriptional control, rather, appeared to be controlled through a negative regulator, nm23, whose own expression was regulated by hypermethylation. Although our studies involved a highly experimental system, the results further suggest that by whatever mechanism, reduction of GnT-V activity through 5-Aza-dC treatment might provide a new approach towards prevention of metastatic progression.

Blocking a novel 55 kDa melanoma-associated cell surface antigen inhibits the development of spontaneous metastases and interactions with frozen lung section.

We recently identified a novel 55-kDa cell-cell adhesion protein (p55) whose expression is upregulated in primary melanomas in the transition from radial growth phase to vertical growth phase. However, the functional role of p55 in various steps of the metastatic process had not been investigated. We provide evidence that subcutaneous injection of metastatic melanoma variant T1P26 in immunosuppressed newborn rats rapidly caused spontaneous metastatic lung lesions that could be readily detected by histochemical analysis with the anti-p55 monoclonal antibody (MAb) LY1. Subsequently, we were able to demonstrate that multiple subcutaneous injections of the LY1 MAb starting on the same day after tumor cell inoculation of T1P26 cells specifically blocked the formation of spontaneous lung metastases, yet had no effects on primary tumor growth, suggesting a critical role of p55 in the earlier steps of the intravasation process. To study later stages in spontaneous metastasis, we investigated the role of p55 in organ-specific cell adhesion of tumor cells in vitro. We showed that the T1P26 variant attached preferentially to lung frozen sections compared with other organs, reflecting the pattern of organ involvement of metastasis in vivo and that LY1 significantly blocked this interaction. However, no significant differences in attachment to lung sections were observed between the parental melanoma cell line M(4)Beu and its derived variant, although cellular topography analysis indicated a preferential attachment of a T1P26 variant on specific compartments of the lungs such as the perialveolar components, the endothelium and the vessel lumen of pulmonary venules. Attachment of the T1P26 variant to lung sections is not due to alterations of tumor cell adherence to basement membrane matrix by the LY1 MAb, suggesting that p55 is involved in cellular adhesion with cellular elements of the lung. p55 could represent a new functional constituent that contributes to the metastatic spread of melanoma cells by promoting the intravasation process and subsequent specific interactions between tumor cells and the target lung organ.

Functional localization of a melanoma tumor suppressor gene to a small (< or = 2 Mb) region on 11q23.

We have previously demonstrated the existence of a melanoma tumor suppressor gene(s) on the long arm of chromosome 11 through suppression of tumorigenicity assays. Although loss of heterozygosity studies also support this finding, only a large critical region (44 cM) has been identified to date on 11q22-25. To further localize a tumor suppressor gene(s) within this region, we have now generated and characterized nine melanoma microcell hybrids, each retaining an introduced fragment of 11q. Of the nine hybrids, four were suppressed for tumor formation in nude mice, while five formed tumors at the same rate as the parental melanoma cell line (UACC 903). Molecular analysis of the hybrids with 118 microsatellite markers narrowed the location of a putative suppressor gene to a small (< or =2 Mb) candidate region on 11q23 between the markers D11S1786 and D11S2077 and within the larger region frequently deleted in melanoma tumors and cell lines. While multiple tumor suppressor genes are likely to reside on 11q22-25, the presence of this region in all four suppressed hybrids supports the simplest model that a single locus is responsible for the suppressed phenotype observed in UACC 903.

Recognition by human Vgamma9/Vdelta2 T cells of melanoma cells upon fusion with Daudi cells.

Daudi Burkitt's lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi x MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vgamma9/Vdelta2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi x melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the gamma/delta T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi x melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vgamma9/Vdelta2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level.