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    The Roles of Extracellular Vesicles in Malignant Melanoma. Cheng Ying-Chen,Chang Yu-An,Chen Yi-Jen,Sung Hsu-Min,Bogeski Ivan,Su Hong-Lin,Hsu Ya-Ling,Wang Hui-Min David Cells Different types of cells, such as endothelial cells, tumor-associated fibroblasts, pericytes, and immune cells, release extracellular vesicles (EVs) in the tumor microenvironment. The components of EVs include proteins, DNA, RNA, and microRNA. One of the most important functions of EVs is the transfer of aforementioned bioactive molecules, which in cancer cells may affect tumor growth, progression, angiogenesis, and metastatic spread. Furthermore, EVs affect the presentation of antigens to immune cells via the transfer of nucleic acids, peptides, and proteins to recipient cells. Recent studies have also explored the potential application of EVs in cancer treatment. This review summarizes the mechanisms by which EVs regulate melanoma development, progression, and their potentials to be applied in therapy. We initially describe vesicle components; discuss their effects on proliferation, anti-melanoma immunity, and drug resistance; and finally focus on the effects of EV-derived microRNAs on melanoma pathobiology. This work aims to facilitate our understanding of the influence of EVs on melanoma biology and initiate ideas for the development of novel therapeutic strategies. 10.3390/cells10102740
    Role of miRNAs in Melanoma Metastasis. Gajos-Michniewicz Anna,Czyz Malgorzata Cancers Tumour metastasis is a multistep process. Melanoma is a highly aggressive cancer and metastasis accounts for the majority of patient deaths. microRNAs (miRNAs) are non-coding RNAs that affect the expression of their target genes. When aberrantly expressed they contribute to the development of melanoma. While miRNAs can act locally in the cell where they are synthesized, they can also influence the phenotype of neighboring melanoma cells or execute their function in the direct tumour microenvironment by modulating ECM (extracellular matrix) and the activity of fibroblasts, endothelial cells, and immune cells. miRNAs are involved in all stages of melanoma metastasis, including intravasation into the lumina of vessels, survival during circulation in cardiovascular or lymphatic systems, extravasation, and formation of the pre-metastatic niche in distant organs. miRNAs contribute to metabolic alterations that provide a selective advantage during melanoma progression. They play an important role in the development of drug resistance, including resistance to targeted therapies and immunotherapies. Distinct profiles of miRNA expression are detected at each step of melanoma development. Since miRNAs can be detected in liquid biopsies, they are considered biomarkers of early disease stages or response to treatment. This review summarizes recent findings regarding the role of miRNAs in melanoma metastasis. 10.3390/cancers11030326
    Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation. Li Wei,Hong Ru,Lai Lan-Tian,Dong Qiman,Ni Peiling,Chelliah Rosi,Huq Mehnaz,Ismail Siti Nadirah Binte,Chandola Udita,Ang Zhiwei,Lin Bingqing,Chen Xin,Chen Lingyi,Zhang Li-Feng Journal of molecular biology Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation. 10.1016/j.jmb.2018.05.031
    Overexpression of DDX43 mediates MEK inhibitor resistance through RAS Upregulation in uveal melanoma cells. Molecular cancer therapeutics The majority of uveal melanomas carry oncogenic mutations in the G proteins GNAQ and GNA11, with consequent activation of the MAPK pathway. Selective MEK inhibitors, such as selumetinib, have shown clinical benefit in uveal melanoma. However, mechanisms of drug resistance limit their efficacy in some patients. Analysis of MEK inhibitor-resistant uveal melanoma cell lines revealed the induction of RAS protein expression and activity. This effect was mediated by the RNA helicase DDX43, which was remarkably overexpressed in these cells. Depletion of DDX43 in MEK inhibitor-resistant cells decreased RAS proteins and inhibited ERK and AKT pathways. On the contrary, ectopic expression of DDX43 in parental uveal melanoma cells induced RAS protein levels and rendered cells resistant to MEK inhibition. Similar to DDX43 depletion, downregulation of KRAS, HRAS, and NRAS inhibited downstream pathways in the resistant cells, overcoming mutant GNAQ signaling. We also analyzed the expression of DDX43 in liver metastases of patients with uveal melanoma by RT-PCR, and found a significant overexpression of DDX43 in patients who did not benefit from selumetinib therapy. In conclusion, DDX43 induces RAS protein expression and signaling, mediating a novel mechanism of MEK inhibitor resistance. The detection of DDX43 in patients with uveal melanoma could lead to more targeted therapies for this disease. 10.1158/1535-7163.MCT-14-0095
    Nanoparticle-delivered transforming growth factor-β siRNA enhances vaccination against advanced melanoma by modifying tumor microenvironment. Xu Zhenghong,Wang Yuhua,Zhang Lu,Huang Leaf ACS nano Achievement of potent immunoresponses against self/tumor antigens and effective therapeutic outcome against advanced tumors remain major challenges in cancer immunotherapy. The specificity and efficiency of two nanoparticle-based delivery systems, lipid-calcium-phosphate (LCP) nanoparticle (NP) and liposome-protamine-hyaluronic acid (LPH) NP, provide us an opportunity to address both challenges. A mannose-modified LCP NP delivered both tumor antigen (Trp 2 peptide) and adjuvant (CpG oligonucleotide) to the dendritic cells and elicited a potent, systemic immune response regardless of the existence or the stage of tumors in the host. This vaccine was less effective, however, against later stage B16F10 melanoma in a subcutaneous syngeneic model. Mechanistic follow-up studies suggest that elevated levels of immune-suppressive cytokines within the tumor microenvironment, such as TGF-β, might be responsible. We strategically augment the efficacy of LCP vaccine on an advanced tumor by silencing TGF-β in tumor cells. The delivery of siRNA using LPH NP resulted in about 50% knockdown of TGF-β in the late stage tumor microenvironment. TGF-β down-regulation boosted the vaccine efficacy and inhibited tumor growth by 52% compared with vaccine treatment alone, as a result of increased levels of tumor infiltrating CD8+ T cells and decreased level of regulatory T cells. Combination of systemic induction of antigen-specific immune response with LCP vaccine and targeted modification of tumor microenvironment with LPH NP offers a flexible and powerful platform for both mechanism study and immunotherapeutic strategy development. 10.1021/nn500216y
    Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma. Falletta Paola,Sanchez-Del-Campo Luis,Chauhan Jagat,Effern Maike,Kenyon Amy,Kershaw Christopher J,Siddaway Robert,Lisle Richard,Freter Rasmus,Daniels Matthew J,Lu Xin,Tüting Thomas,Middleton Mark,Buffa Francesca M,Willis Anne E,Pavitt Graham,Ronai Ze'ev A,Sauka-Spengler Tatjana,Hölzel Michael,Goding Colin R Genes & development The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome. 10.1101/gad.290940.116
    ERK-mediated phosphorylation regulates SOX10 sumoylation and targets expression in mutant BRAF melanoma. Han Shujun,Ren Yibo,He Wangxiao,Liu Huadong,Zhi Zhe,Zhu Xinliang,Yang Tielin,Rong Yu,Ma Bohan,Purwin Timothy J,Ouyang Zhenlin,Li Caixia,Wang Xun,Wang Xueqiang,Yang Huizi,Zheng Yan,Aplin Andrew E,Liu Jiankang,Shao Yongping Nature communications In human mutant BRAF melanoma cells, the stemness transcription factor FOXD3 is rapidly induced by inhibition of ERK1/2 signaling and mediates adaptive resistance to RAF inhibitors. However, the mechanism underlying ERK signaling control of FOXD3 expression remains unknown. Here we show that SOX10 is both necessary and sufficient for RAF inhibitor-induced expression of FOXD3 in mutant BRAF melanoma cells. SOX10 activates the transcription of FOXD3 by binding to a regulatory element in FOXD3 promoter. Phosphorylation of SOX10 by ERK inhibits its transcription activity toward multiple target genes by interfering with the sumoylation of SOX10 at K55, which is essential for its transcription activity. Finally, depletion of SOX10 sensitizes mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo. Thus, our work discovers a novel phosphorylation-dependent regulatory mechanism of SOX10 transcription activity and completes an ERK1/2/SOX10/FOXD3/ERBB3 axis that mediates adaptive resistance to RAF inhibitors in mutant BRAF melanoma cells. 10.1038/s41467-017-02354-x
    Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing. Salton Maayan,Kasprzak Wojciech K,Voss Ty,Shapiro Bruce A,Poulikakos Poulikos I,Misteli Tom Nature communications Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma. 10.1038/ncomms8103
    The lncRNA SLNCR1 Mediates Melanoma Invasion through a Conserved SRA1-like Region. Schmidt Karyn,Joyce Cailin E,Buquicchio Frank,Brown Adam,Ritz Justin,Distel Robert J,Yoon Charles H,Novina Carl D Cell reports Long non-coding RNAs (lncRNAs) have been implicated in numerous physiological processes and diseases, most notably cancers. However, little is known about the mechanism of many functional lncRNAs. We identified an abundantly expressed lncRNA associated with decreased melanoma patient survival. Increased expression of this lncRNA, SLNCR1, mediates melanoma invasion through a highly conserved sequence similar to that of the lncRNA SRA1. Using a sensitive technique we term RATA (RNA-associated transcription factor array), we show that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bind within and adjacent to SLNCR1's conserved region, respectively. SLNCR1, AR, and Brn3a are specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. Our observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival in comparison to females. 10.1016/j.celrep.2016.04.018
    ADAR1-mediated regulation of melanoma invasion. Nemlich Yael,Baruch Erez Nissim,Besser Michal Judith,Shoshan Einav,Bar-Eli Menashe,Anafi Liat,Barshack Iris,Schachter Jacob,Ortenberg Rona,Markel Gal Nature communications Melanoma cells use different migratory strategies to exit the primary tumor mass and invade surrounding and subsequently distant tissues. We reported previously that ADAR1 expression is downregulated in metastatic melanoma, thereby facilitating proliferation. Here we show that ADAR1 silencing enhances melanoma cell invasiveness and ITGB3 expression. The enhanced invasion is reversed when ITGB3 is blocked with antibodies. Re-expression of wild-type or catalytically inactive ADAR1 establishes this mechanism as independent of RNA editing. We demonstrate that ADAR1 controls ITGB3 expression both at the post-transcriptional and transcriptional levels, via miR-22 and PAX6 transcription factor, respectively. These are proven here as direct regulators of ITGB3 expression. miR-22 expression is controlled by ADAR1 via FOXD1 transcription factor. Clinical relevance is demonstrated in patient-paired progression tissue microarray using immunohistochemistry. The novel ADAR1-dependent and RNA-editing-independent regulation of invasion, mediated by ITGB3, strongly points to a central involvement of ADAR1 in cancer progression and metastasis. 10.1038/s41467-018-04600-2
    An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells. Shen Shensi,Faouzi Sara,Bastide Amandine,Martineau Sylvain,Malka-Mahieu Hélène,Fu Yu,Sun Xiaoxiao,Mateus Christine,Routier Emilie,Roy Severine,Desaubry Laurent,André Fabrice,Eggermont Alexander,David Alexandre,Scoazec Jean-Yves,Vagner Stéphan,Robert Caroline Nature communications Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse. Here we show that a subpopulation of BRAF mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity. Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation. Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells. Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5'-untranslated region of some highly translated mRNAs. Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance. 10.1038/s41467-019-13360-6
    The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma. Phung Bengt,Cieśla Maciej,Sanna Adriana,Guzzi Nicola,Beneventi Giulia,Cao Thi Ngoc Phuong,Lauss Martin,Cabrita Rita,Cordero Eugenia,Bosch Ana,Rosengren Frida,Häkkinen Jari,Griewank Klaus,Paschen Annette,Harbst Katja,Olsson Håkan,Ingvar Christian,Carneiro Ana,Tsao Hensin,Schadendorf Dirk,Pietras Kristian,Bellodi Cristian,Jönsson Göran Cell reports The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5' UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential in vivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas. 10.1016/j.celrep.2019.05.069
    Wild-type p53 gene transfer inhibits invasion and reduces matrix metalloproteinase-2 levels in p53-mutated human melanoma cells. Toschi E,Rota R,Antonini A,Melillo G,Capogrossi M C The Journal of investigative dermatology The tumor suppressor gene p53 has inhibitory effects on cell growth and angiogenesis and induces apoptosis when overexpressed in melanoma and in a variety of tumor cells by adenovirus-mediated gene transfer. The invasive ability of tumor cells, facilitating local infiltration and metastasis, is related to matrix metalloproteinase levels. In melanoma, matrix metalloproteinase-2 and matrix metalloproteinase-9 have a prominent role in this process. The aim of this study was to evaluate whether wild-type p53 overexpression, obtained by a recombinant adenovirus vector (AdCMV.p53), affects cell invasiveness through modulation of matrix metalloproteinase-2 and matrix metalloproteinase-9. Two human melanoma cell lines were used in this study: the SK-MEL-110, carrying a mutated p53 gene, and the SK-MEL-147, carrying the wild-type p53 gene. SK-MEL-110 cells infected with AdCMV.p53 exhibited decreased invasion capability from day 1 after infection, compared with cells not infected or infected with the control vector AdCMV.Null. This reduced invasiveness was associated with decreased matrix metalloproteinase-2 levels in conditioned media whereas no changes were detected in matrix metalloproteinase-9 secreted levels. No modulation in matrix metalloproteinase-2 mRNA levels was detectable, however, after wild-type p53 gene transfer. Furthermore, protein expression of secreted tissue inhibitor of metalloproteinase-2 was not altered by AdCMV.p53 treatment. In contrast, in SK-MEL-147 cells, AdCMV.p53 did not affect cell invasiveness and levels of secreted matrix metalloproteinase-2. Gene transfer of wild-type p53 inhibited proliferation of both cell lines, showing that also SK-MEL-147 cells respond to wild-type p53 overexpression. This novel mechanism of action of wild-type p53 gene transfer may contribute to its antitumor effect by downregulating cell invasion and matrix metalloproteinase-2 secreted levels in mutated p53 human melanoma cell lines. 10.1046/j.1523-1747.2000.00000.x
    Comparison of d-g3139 and its enantiomer L-g3139 in melanoma cells demonstrates minimal in vitro but dramatic in vivo chiral dependency. Lai Johnathan C,Brown Bob D,Voskresenskiy Anatoliy M,Vonhoff Stefan,Klussman Sven,Tan Wenzhi,Colombini Marco,Weeratna Risini,Miller Paul,Benimetskaya Luba,Stein Cy A Molecular therapy : the journal of the American Society of Gene Therapy G3139 (Genasense), an 18mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 messenger RNA (mRNA), downregulates Bcl-2 protein and mRNA expression in many cell lines. However, both the in vitro and in vivo mechanisms of action of G3139 are still uncertain. The isosequential L-deoxyribose enantiomer L-G3139, which does not downregulate Bcl-2 expression, was synthesized to study the role of the Bcl-2 protein in melanoma cells. Both D-G3139 and L-G3139 bind nonspecifically to basic fibroblast growth factor with approximately the same K(c), and cause highly effective inhibition of net formation in 518A2 melanoma cells on Matrigel. The uptakes of D-G3139 and L-G3139 in melanoma cells were also similar. However, unlike D-G3139, L-G3139 does not produce poly ADP-ribose polymerase-1 and procaspase-3 cleavage at 9.5 h after the initiation of the transfection, but can activate the intrinsic pathway of apoptosis at approximately 48 h. Furthermore, treatment of A375 melanoma human xenografts in severe combined immunodeficiency (SCID) mice demonstrates that tumor growth is not inhibited by L-G3139, whereas D-G3139 significantly inhibits the rate of tumor growth. Furthermore, the immunostimulatory properties of L-G3139 appear to be nil, which differs dramatically from those of D-G3139. In conclusion, profound differences exist between D-G3139 and L-G3139 in vivo despite their similarities in vitro. 10.1038/sj.mt.6300037
    Oncogenic BRAF is required for tumor growth and maintenance in melanoma models. Hoeflich Klaus P,Gray Daniel C,Eby Michael T,Tien Janet Y,Wong Leo,Bower Janeko,Gogineni Alvin,Zha Jiping,Cole Mary J,Stern Howard M,Murray Lesley J,Davis David P,Seshagiri Somasekar Cancer research The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics. 10.1158/0008-5472.CAN-05-2720
    TIGIT-CD155 Interactions in Melanoma: A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention. Mahnke Karsten,Enk Alexander H The Journal of investigative dermatology Inozume et al. describe a novel immunosuppressive mechanism in melanoma that is triggered by the interaction between CD155 (expressed by melanomas) and T-cell Ig and ITIM domain (TIGIT) (expressed by tumor infiltrating lymphocytes). This pathway exists in addition to the "classical" co-inhibitory PD-1-PD-L1 pathway. Hence, the combinatorial blockage of both pathways by specific antibodies resulted in the greatly enhanced effector function of melanoma-specific cytotoxic T cells. Given that CD155-TIGIT signaling exerts potent inhibitory action in different subsets of immune cells and that CD155 is expressed broadly in several tumor entities, this report points toward a novel and promising therapeutic strategy to combine different checkpoint blocking agents for greater success in antitumor therapy. 10.1016/j.jid.2015.10.048
    A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species. Verhaegen Monique,Bauer Joshua A,Martín de la Vega Cristina,Wang Guoping,Wolter Keith G,Brenner J Chadwick,Nikolovska-Coleska Zaneta,Bengtson Audrey,Nair Rajan,Elder James T,Van Brocklin Matt,Carey Thomas E,Bradford Carol R,Wang Shaomeng,Soengas María S Cancer research The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design. 10.1158/0008-5472.CAN-06-1748
    Intracellular targets of RGDS peptide in melanoma cells. Aguzzi Maria Simona,Fortugno Paola,Giampietri Claudia,Ragone Gianluca,Capogrossi Maurizio C,Facchiano Antonio Molecular cancer BACKGROUND:RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment. RESULTS:In the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 muM) and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA. CONCLUSIONS:RGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular matrix and may suggest a possible novel anti-proliferation strategy in melanoma. 10.1186/1476-4598-9-84
    Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells. Das Swadesh K,Sokhi Upneet K,Bhutia Sujit K,Azab Belal,Su Zhao-Zhong,Sarkar Devanand,Fisher Paul B Proceedings of the National Academy of Sciences of the United States of America MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3'-5' exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase(old-35) identified miRNAs differentially and specifically regulated by hPNPase(old-35). One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27(kip1), displayed robust down-regulation with ensuing up-regulation of p27(kip1) by expression of hPNPase(old-35), which also occurred in multiple human melanoma cells upon IFN-beta treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase(old-35). Inhibition of hPNPase(old-35) by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-beta-mediated growth inhibition, accentuating the importance of hPNPase(old-35) induction and miR-221 down-regulation in mediating IFN-beta action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase(old-35) might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma. 10.1073/pnas.0914143107
    The abrogation of the HOXB7/PBX2 complex induces apoptosis in melanoma through the miR-221&222-c-FOS pathway. Errico M Cristina,Felicetti Federica,Bottero Lisabianca,Mattia Gianfranco,Boe Alessandra,Felli Nadia,Petrini Marina,Bellenghi Maria,Pandha Hardev S,Calvaruso Marco,Tripodo Claudio,Colombo Mario P,Morgan Richard,Carè Alessandra International journal of cancer Cutaneous melanoma is the fastest increasing cancer worldwide. Although several molecular abnormalities have been associated with melanoma progression, the underlying mechanisms are still largely unknown and few targeted therapies are under evaluation. Here we show that the HOXB7/PBX2 dimer acts as a positive transcriptional regulator of the oncogenic microRNA-221 and -222. In addition, demonstrating c-FOS as a direct target of miR-221&222, we identify a HOXB7/PBX2→miR-221&222 →c-FOS regulatory link, whereby the abrogation of functional HOXB7/PBX2 dimers leads to reduced miR-221&222 transcription and elevated c-FOS expression with consequent cell death. Taking advantage of the treatment with the peptide HXR9, an antagonist of HOX/PBX dimerization, we recognize miR-221&222 as effectors of its action, in turn confirming the HXR9 efficacy in the treatment of human melanoma malignancy, whilst sparing normal human melanocytes. Our findings, besides suggesting the potential therapeutic of HXR9 or its derivatives in malignant melanoma, suggest the disruption of the HOXB7/PBX2 complexes, miR-221&222 inhibition or even better their combination, as innovative therapeutic approaches. 10.1002/ijc.28097
    The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression. Dar Altaf A,Majid Shahana,Rittsteuer Claudia,de Semir David,Bezrookove Vladimir,Tong Schuyler,Nosrati Mehdi,Sagebiel Richard,Miller James R,Kashani-Sabet Mohammed Journal of the National Cancer Institute BACKGROUND:Although p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene. Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway. METHODS:Expression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes. Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b. The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group). Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing. RESULTS:Expression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment. MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition. CONCLUSIONS:Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors. 10.1093/jnci/djt003
    RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nature cell biology The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(aln) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress. 10.1038/s41556-020-0493-0
    Antitumor activity of miR-1280 in melanoma by regulation of Src. Sun Vera,Zhou Wen B,Nosrati Mehdi,Majid Shahana,Thummala Suresh,de Semir David,Bezrookove Vladimir,de Feraudy Sebastien,Chun Liane,Schadendorf Dirk,Debs Robert,Kashani-Sabet Mohammed,Dar Altaf A Molecular therapy : the journal of the American Society of Gene Therapy MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3' untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma. 10.1038/mt.2014.176
    The mucin protein MUCL1 regulates melanogenesis and melanoma genes in a manner dependent on threonine content. The British journal of dermatology BACKGROUND:The regulation of melanogenesis has been investigated as a long-held aim for pharmaceutical manipulations with denotations for malignancy of melanoma. Mucins have a protective function in epithelial organs; however, in the most outer organ, the skin, the role of mucins has not been studied enough. OBJECTIVES:Our initial hypothesis developed from the identification of correlations between pigmentation and expressions of skin mucins, particularly those existing in skin tissue. We aimed to investigate the action of mucins in human melanocytic cells. MATERIALS AND METHODS:The expression of mucin proteins in human skin was investigated using microarray data from the Human Protein Atlas consortium (HPA) and the Genotype-Tissue Expression consortium (GTEx) database. Mucin expression was measured at RNA and protein levels in melanoma cells. The findings were further validated and confirmed by analysis of independent experiments. RESULTS:We found that the several mucin proteins showed expression in human skin cells and among these, mucin-like protein 1 (MUCL1) showed the highest expression and also clear negative correlation with melanogenesis in epidermal melanocytes. We confirmed the correlations between melanogenesis and MUCL1 by revealing negative correlations in melanocytes with different melanin production, resulting from increased composition of threonine, mucin-conforming amino acid, and increased autophagy-related forkhead-box O signalling. Furthermore, threonine itself affects melanogenesis and metastatic activity in melanoma cells. CONCLUSIONS:We identified a significant association between MUCL1 and threonine with melanogenesis and metastasis-related genes in melanoma cells. Our results define a novel mechanism of mucin regulation, suggesting diagnostic and preventive roles of MUCL1 in cutaneous melanoma. 10.1111/bjd.20761
    Squaring the circle: circRNAs in melanoma. Mecozzi Nicol,Vera Olga,Karreth Florian A Oncogene Non-coding RNAs are emerging as critical molecules in the genesis, progression, and therapy resistance of cutaneous melanoma. This includes circular RNAs (circRNAs), a class of non-coding RNAs with distinct characteristics that forms through non-canonical back-splicing. In this review, we summarize the features and functions of circRNAs and introduce the current knowledge of the roles of circRNAs in melanoma. We also highlight the various mechanisms of action of the well-studied circRNA CDR1as and describe how it acts as a melanoma tumor suppressor. We further discuss the utility of circRNAs as biomarkers, therapeutic targets, and therapeutic agents in melanoma and outline challenges that must be overcome to comprehensively characterize circRNA functions. 10.1038/s41388-021-01977-1
    Joint action of miR-126 and MAPK/PI3K inhibitors against metastatic melanoma. Pedini Francesca,De Luca Gabriele,Felicetti Federica,Puglisi Rossella,Boe Alessandra,Arasi Maria Beatrice,Fratini Federica,Mattia Gianfranco,Spada Massimo,Caporali Simona,Biffoni Mauro,Giuliani Alessandro,Carè Alessandra,Felli Nadia Molecular oncology Emerging data support the rationale of combined therapies in advanced melanoma. Specifically, the combined use of drugs with different mechanisms of action can reduce the probability of selecting resistant clones. To identify agents active against melanoma cells, we screened a library of 349 anti-cancer compounds, currently in clinical use or trials, and selected PIK-75, an inhibitor of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, as the 'top active' drug. PIK-75 was then used alone or in combination with vemurafenib, the first BRAF inhibitor approved for patients with melanoma harboring BRAF mutations. We identified a combined dose of PIK-75 and vemurafenib that inhibited both the PI3K/AKT and mitogen-activated protein kinase pathways, thereby overcoming any compensatory activation. In view of the important tumor suppressor function induced by restoring expression of microRNA (miR)-126 in metastatic melanoma cells, we examined whether miR-126 has a synergistic role when included in a triple combination alongside PIK-75 and vemurafenib. We found that enforced expression of miR-126 (which alone can reduce tumorigenicity) significantly increased PIK-75 activity when used as either a single agent or in combination with vemurafenib. Interestingly, PIK-75 proved to be effective against early passage cell lines derived from patients' biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, thus suggesting that it potentially has the capability to overcome drug resistance. Finally, the synergistic role played by miR-126 in combination with vemurafenib and/or PIK-75 was demonstrated in vivo in mouse xenograft models, in which tumor growth inhibition was associated with increased apoptosis. These results not only show the efficacy of PIK-75 and vemurafenib co-treatment but also indicate that restoration of miR-126 expression in advanced melanoma can enhance their antitumor activity, which may possibly allow dose reduction to decrease adverse events without reducing the therapeutic benefits. 10.1002/1878-0261.12506
    MALAT1 regulates miR-34a expression in melanoma cells. Li Fei,Li Xinji,Qiao Li,Liu Wen,Xu Chengshan,Wang Xiaogang Cell death & disease Melanoma is one of the most common skin malignancies. Both microRNAs and long non-coding RNAs (lncRNAs) have critical roles in the progression of cancers, including melanoma. However, the underlying molecular mechanism has not been fully characterized. We demonstrated that miR-34a is negatively correlated with MALAT1 in melanoma cells and tumor specimens. Interestingly, MALAT1, which contains functional sequence-specific miR-34a-binding sites, regulates miR-34a stability in melanoma cells and in vivo. Importantly, MALAT1 was significantly enriched in the Ago2 complex, but not when the MALAT1-binding site of miR-34a was mutated. Furthermore, MALAT1 could be shown to regulate c-Myc and Met expression by functioning as a miR-34a sponge. Our results reveal an unexpected mode of action for MALAT1 as an important regulator of miR-34a. 10.1038/s41419-019-1620-3
    Polymorphisms in melanoma differentiation-associated gene 5 link protein function to clearance of hepatitis C virus. Hoffmann Franziska S,Schmidt Andreas,Dittmann Chevillotte Meike,Wisskirchen Christian,Hellmuth Johannes,Willms Simone,Gilmore Rachel H,Glas Jürgen,Folwaczny Matthias,Müller Tobias,Berg Thomas,Spengler Ulrich,Fitzmaurice Karen,Kelleher Dermot,Reisch Nicole,Rice Charles M,Endres Stefan,Rothenfusser Simon Hepatology (Baltimore, Md.) UNLABELLED:Among patients newly infected with hepatitis C virus (HCV), only 20-30% clear the infection spontaneously. In the remaining 70% the infection persists, causing chronic liver inflammation and disease. It is well established that polymorphisms in host genes, especially in components of the innate immune response, contribute to the phenomenon of spontaneous HCV clearance. Retinoic acid inducible gene-I (RIG-I)-like helicases such as melanoma differentiation-associated gene 5 (MDA-5) are cytoplasmic sensors of viral RNA that are critical for triggering innate immune responses after infection with RNA viruses. We analyzed 14 nonsynonymous single-nucleotide polymorphisms in RIG-I-like helicase-pathway-genes comparing European patients who spontaneously cleared HCV (n = 285) or had persistent infection (n = 509). We found that polymorphic haplotypes in the MDA-5 gene IFIH1 encoding histidine at position 843 and threonine at position 946 strongly correlate with the resolution of HCV infection (odds ratio [OR]: 16.23; 95% confidence interval [CI]: 3.67-71.87; P = 1.1 × 10(-6) ). Overexpression of MDA-5 genetic variants in HEK 293 cells and in a tissue culture model of HCV infection revealed that the histidine 843/threonine 946 variant leads to increased baseline and ligand-induced expression of interferon-induced genes and confers an increased ability to suppress HCV replication. CONCLUSION:These data suggest that MDA-5 plays a significant role in the defense against HCV and that polymorphisms in MDA-5 can influence the outcome of HCV infection. 10.1002/hep.27344
    Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses. Chiappinelli Katherine B,Strissel Pamela L,Desrichard Alexis,Li Huili,Henke Christine,Akman Benjamin,Hein Alexander,Rote Neal S,Cope Leslie M,Snyder Alexandra,Makarov Vladimir,Budhu Sadna,Buhu Sadna,Slamon Dennis J,Wolchok Jedd D,Pardoll Drew M,Beckmann Matthias W,Zahnow Cynthia A,Merghoub Taha,Mergoub Taha,Chan Timothy A,Baylin Stephen B,Strick Reiner Cell We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model. 10.1016/j.cell.2015.07.011
    The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells. Sumimoto Hidetoshi,Imabayashi Fumie,Iwata Tomoko,Kawakami Yutaka The Journal of experimental medicine The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAF(V600E)). MEK inhibitor U0126 or RNA interference (RNAi) for BRAF(V600E) decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor alpha by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAF(V600E) RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAF(V600E) and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system. 10.1084/jem.20051848
    Targeting Adenosine in BRAF-Mutant Melanoma Reduces Tumor Growth and Metastasis. Young Arabella,Ngiow Shin Foong,Madore Jason,Reinhardt Julia,Landsberg Jennifer,Chitsazan Arash,Rautela Jai,Bald Tobias,Barkauskas Deborah S,Ahern Elizabeth,Huntington Nicholas D,Schadendorf Dirk,Long Georgina V,Boyle Glen M,Hölzel Michael,Scolyer Richard A,Smyth Mark J Cancer research Increasing evidence exists for the role of immunosuppressive adenosine in promoting tumor growth and spread in a number of cancer types, resulting in poor clinical outcomes. In this study, we assessed whether the CD73-adenosinergic pathway is active in melanoma patients and whether adenosine restricts the efficacy of clinically approved targeted therapies for commonly mutated BRAF melanoma. In AJCC stage III melanoma patients, CD73 expression (the enzyme that generates adenosine) correlated significantly with patients presenting nodal metastatic melanoma, suggesting that targeting this pathway may be effective in advanced stage disease. In addition, dabrafenib and trametinib treatment of CD73 BRAF-mutant melanomas caused profound CD73 downregulation in tumor cells. Inhibition of BRAF and MEK in combination with the A2A adenosine receptor provided significant protection against tumor initiation and metastasis formation in mice. Our results suggest that targeting adenosine may enhance therapeutic responses for melanoma patients receiving targeted or immune-based therapies. . 10.1158/0008-5472.CAN-17-0393
    Interferon-γ Signaling in Melanocytes and Melanoma Cells Regulates Expression of CTLA-4. Mo Xuan,Zhang Hanghang,Preston Sarah,Martin Kayla,Zhou Bo,Vadalia Nish,Gamero Ana M,Soboloff Jonathan,Tempera Italo,Zaidi M Raza Cancer research CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T-cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here, we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-γ (IFNG) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNG activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNG-response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. Taken together, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses. These findings show that human melanoma cells express high levels of the immune checkpoint molecule CTLA4, with important possible implications for understanding how anti-CTLA4 immunotherapy mediates its therapeutic effects. . 10.1158/0008-5472.CAN-17-1615
    Abl kinase regulation by BRAF/ERK and cooperation with Akt in melanoma. Jain A,Tripathi R,Turpin C P,Wang C,Plattner R Oncogene The melanoma incidence continues to increase, and the disease remains incurable for many due to its metastatic nature and high rate of therapeutic resistance. In particular, melanomas harboring BRAF and PTEN mutations often are resistant to current therapies, including BRAF inhibitors (BRAFi) and immune checkpoint inhibitors. Abl kinases (Abl/Arg) are activated in melanomas and drive progression; however, their mechanism of activation has not been established. Here we elucidate a novel link between BRAF/ERK signaling and Abl kinases. We demonstrate that BRAF/ERK play a critical role in binding, phosphorylating and regulating Abl localization and Abl/Arg activation by Src family kinases. Importantly, Abl/Arg activation downstream of BRAF has functional and biological significance, driving proliferation, invasion, as well as switch in epithelial-mesenchymal-transition transcription factor expression, which is known to be critical for melanoma cells to shift between differentiated and invasive states. Finally, we describe findings of high translational significance by demonstrating that Abl/Arg cooperate with PI3K/Akt/PTEN, a parallel pathway that is associated with intrinsic resistance to BRAFi and immunotherapy, as Abl/Arg and Akt inhibitors cooperate to prevent viability, cell cycle progression and in vivo growth of melanomas harboring mutant BRAF/PTEN. Thus, these data not only provide mechanistic insight into Abl/Arg regulation during melanoma development, but also pave the way for the development of new strategies for treating patients with melanomas harboring mutant BRAF/PTEN, which often are refractory to current therapies. 10.1038/onc.2017.76
    Metabolic therapy with PEG-arginase induces a sustained complete remission in immunotherapy-resistant melanoma. De Santo Carmela,Cheng Paul,Beggs Andrew,Egan Sharon,Bessudo Alberto,Mussai Francis Journal of hematology & oncology BACKGROUND:Metastatic melanoma is an aggressive skin cancer with a poor prognosis. Current treatment strategies for high-stage melanoma are based around the use of immunotherapy with immune checkpoint inhibitors such as anti-PDL1 or anti-CTLA4 antibodies to stimulate anti-cancer T cell responses, yet a number of patients will relapse and die of disease. Here, we report the first sustained complete remission in a patient with metastatic melanoma who failed two immunotherapy strategies, by targeting tumour arginine metabolism. CASE PRESENTATION:A 65-year-old patient with metastatic melanoma who progressed through two immunotherapy strategies with immune checkpoint inhibitor antibodies was enrolled in a phase I study (NCT02285101) and treated with 2 mg/kg intravenously, weekly pegylated recombinant arginase (BCT-100). The patient experienced no toxicities > grade 2 and entered a complete remission which is sustained for over 30 months. RNA-sequencing identified a number of transcriptomic pathway alterations compared to control samples. The tumour had absent expression of the recycling enzymes argininosuccinate synthetase (ASS) and ornithine transcarbamylase (OTC) indicating a state of arginine auxotrophy, which was reconfirmed by immunohistochemistry, and validation in a larger cohort of melanoma tumour samples. CONCLUSIONS:Targeting arginine metabolism with therapeutic arginase in arginine auxotrophic melanoma can be an effective salvage for the treatment of patients who fail immunotherapy. 10.1186/s13045-018-0612-6
    Adjuvant dabrafenib plus trametinib versus placebo in patients with resected, BRAF-mutant, stage III melanoma (COMBI-AD): exploratory biomarker analyses from a randomised, phase 3 trial. Dummer Reinhard,Brase Jan C,Garrett James,Campbell Catarina D,Gasal Eduard,Squires Matthew,Gusenleitner Daniel,Santinami Mario,Atkinson Victoria,Mandalà Mario,Chiarion-Sileni Vanna,Flaherty Keith,Larkin James,Robert Caroline,Kefford Richard,Kirkwood John M,Hauschild Axel,Schadendorf Dirk,Long Georgina V The Lancet. Oncology BACKGROUND:Adjuvant dabrafenib plus trametinib reduced the risk of relapse versus placebo in patients with resected, BRAF-mutant, stage III melanoma in the phase 3 COMBI-AD trial. This prespecified exploratory biomarker analysis aimed to evaluate potential prognostic or predictive factors and mechanisms of resistance to adjuvant targeted therapy. METHODS:COMBI-AD is a randomised, double-blind, placebo-controlled, phase 3 trial comparing dabrafenib 150 mg orally twice daily plus trametinib 2 mg orally once daily versus two matched placebos. Study participants were at least 18 years of age and underwent complete resection of stage IIIA (lymph node metastases >1 mm), IIIB, or IIIC cutaneous melanoma, per American Joint Committee on Cancer 7th edition criteria, with a BRAF or BRAF mutation. Patients were randomly assigned (1:1) to the two treatment groups by an interactive voice response system, stratified by mutation type and disease stage. Patients, physicians, and the investigators who analysed data were masked to treatment allocation. The primary outcome was relapse-free survival, defined as the time from randomisation to disease recurrence or death from any cause. Biomarker assessment was a prespecified exploratory outcome of the trial. We assessed intrinsic tumour genomic features by use of next-generation DNA sequencing and characteristics of the tumour microenvironment by use of a NanoString RNA assay, which might provide prognostic and predictive information. This trial is registered with ClinicalTrials.gov, number NCT01682083, and is ongoing but no longer recruiting participants. FINDINGS:Between Jan 31, 2013, and Dec 11, 2014, 870 patients were enrolled in the trial. Median follow-up at data cutoff (April 30, 2018) was 44 months (IQR 38-49) in the dabrafenib plus trametinib group and 42 months (21-49) in the placebo group. Intrinsic tumour genomic features were assessed in 368 patients (DNA sequencing set) and tumour microenvironment characteristics were assessed in 507 patients (NanoString biomarker set). MAPK pathway genomic alterations at baseline did not affect treatment benefit or clinical outcome. An IFNγ gene expression signature higher than the median was prognostic for prolonged relapse-free survival in both treatment groups. Tumour mutational burden was independently prognostic for relapse-free survival in the placebo group (high TMB, top third; hazard ratio [HR] 0·56, 95% CI 0·37-0·85, p=0·0056), but not in the dabrafenib plus trametinib group (0·83, 95% CI 0·53-1·32, p=0·44). Patients with tumour mutational burden in the lower two terciles seem to derive a substantial long-term relapse-free survival benefit from targeted therapy (HR [versus placebo] 0·49, 95% CI 0·35-0·68, p<0·0001). However, patients with high tumour mutational burden seem to have a less pronounced benefit with targeted therapy (HR [versus placebo] 0·75, 95% CI 0·44-1·26, p=0·27), especially if they had an IFNγ signature lower than the median (HR 0·88 [95% CI 0·40-1·93], p=0·74). INTERPRETATION:Tumour mutational burden alone or in combination with IFNγ gene expression signature or other markers for an adaptive immune response might be of relevance for identifying patients with stage III melanoma who might derive clinical benefit from targeted therapy. Further validation in prospective clinical trials is warranted. FUNDING:Novartis Pharmaceuticals. 10.1016/S1470-2045(20)30062-0
    Anti-tumour immunity induces aberrant peptide presentation in melanoma. Bartok Osnat,Pataskar Abhijeet,Nagel Remco,Laos Maarja,Goldfarb Eden,Hayoun Deborah,Levy Ronen,Körner Pierre-Rene,Kreuger Inger Z M,Champagne Julien,Zaal Esther A,Bleijerveld Onno B,Huang Xinyao,Kenski Juliana,Wargo Jennifer,Brandis Alexander,Levin Yishai,Mizrahi Orel,Alon Michal,Lebon Sacha,Yang Weiwen,Nielsen Morten M,Stern-Ginossar Noam,Altelaar Maarten,Berkers Celia R,Geiger Tamar,Peeper Daniel S,Olweus Johanna,Samuels Yardena,Agami Reuven Nature Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape. 10.1038/s41586-020-03054-1
    The immune microenvironment confers resistance to MAPK pathway inhibitors through macrophage-derived TNFα. Smith Michael P,Sanchez-Laorden Berta,O'Brien Kate,Brunton Holly,Ferguson Jennifer,Young Helen,Dhomen Nathalie,Flaherty Keith T,Frederick Dennie T,Cooper Zachary A,Wargo Jennifer A,Marais Richard,Wellbrock Claudia Cancer discovery UNLABELLED:Recently, the rationale for combining targeted therapy with immunotherapy has come to light, but our understanding of the immune response during MAPK pathway inhibitor treatment is limited. We discovered that the immune microenvironment can act as a source of resistance to MAPK pathway-targeted therapy, and moreover during treatment this source becomes reinforced. In particular, we identified macrophage-derived TNFα as a crucial melanoma growth factor that provides resistance to MAPK pathway inhibitors through the lineage transcription factor MITF (microphthalmia transcription factor). Most strikingly, in BRAF-mutant melanomas of patients and BRAF(V600E) melanoma allografts, MAPK pathway inhibitors increased the number of tumor-associated macrophages, and TNFα and MITF expression. Inhibiting TNFα signaling with IκB kinase inhibitors profoundly enhanced the efficacy of MAPK pathway inhibitors by targeting not only the melanoma cells but also the microenvironment. In summary, we identify the immune microenvironment as a novel source of resistance and reveal a new strategy to improve the efficacy of targeted therapy in melanoma. SIGNIFICANCE:This study identifies the immune microenvironment as a source of resistance to MAPK pathway inhibitors through macrophage-derived TNFα, and reveals that in patients on treatment this source becomes reinforced. Inhibiting IκB kinase enhances the efficacy of MAPK pathway inhibitors, which identifies this approach as a potential novel strategy to improve targeted therapy in melanoma. 10.1158/2159-8290.CD-13-1007
    Inhibition of NOS1 promotes the interferon response of melanoma cells. Journal of translational medicine BACKGROUND:NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown. METHODS:The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer. RESULTS:NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients. CONCLUSION:These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells. 10.1186/s12967-022-03403-w
    Targeting STAT3 Abrogates Tim-3 Upregulation of Adaptive Resistance to PD-1 Blockade on Regulatory T Cells of Melanoma. Huang Lili,Xu Yu,Fang Juemin,Liu Weixing,Chen Jianhua,Liu Zhuqing,Xu Qing Frontiers in immunology Background:Less than 20% of melanoma patients respond to programmed cell death-1 (PD-1) blockade immunotherapies. Thus, it is crucial to understand the dynamic changes in the tumor microenvironment (TME) after PD-1 blockade, for developing immunotherapy efficacy. Methods:A genomic analysis was conducted by The Cancer Genome Atlas (TCGA) datasets and web platform TIMER2.0 datasets. Pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Peripheral blood mononuclear cells (PBMCs), regulatory T (Treg) cells, and B16-F10 melanoma mice were used as models. The cellular and molecular characteristics and mechanisms of Treg cells in melanoma were assessed by performing gene expression studies, immunohistochemistry, RNA sequencing, and flow cytometry. Results:Here, we evaluate the countenance of T cell immunoglobulin and mucin-domain containing-3 (Tim-3), and various immunosuppressive factors within tumor-infiltrated Treg cells after treatment with anti-PD-1 or the indicator transduction and activator of transcription 3 (STAT3) inhibitors. Increased expression of Tim-3 is markedly observed within the tissues of the PD-1 blockade resistance of melanoma patients. Targeting STAT3 significantly boosts the response of resistant-PD-1 therapy within the melanoma mouse model. Mechanistically, the manifestation of STAT3 decreases the expression of Tim-3 and various cytokines in the purified Treg cells from individual PBMCs and the murine melanoma model, limiting the immunosuppression of Treg cells. Conclusions:Our findings indicate that Tim-3 expression on Treg cells within the TME is STAT3-dependent, providing support to STAT3 as a target and enhancing the immunotherapy for patients suffering from melanoma. 10.3389/fimmu.2021.654749
    IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression. Thiem Alexander,Hesbacher Sonja,Kneitz Hermann,di Primio Teresa,Heppt Markus V,Hermanns Heike M,Goebeler Matthias,Meierjohann Svenja,Houben Roland,Schrama David Journal of experimental & clinical cancer research : CR BACKGROUND:Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. METHODS:We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. RESULTS:For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53, a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53 in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. CONCLUSIONS:While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy. 10.1186/s13046-019-1403-9
    New evaluation of the tumor immune microenvironment of non-small cell lung cancer and its association with prognosis. Journal for immunotherapy of cancer BACKGROUND:A better understanding of the tumor immune microenvironment (TIME) will facilitate the development of prognostic biomarkers and more effective therapeutic strategies in patients with lung cancer. However, little has been reported on the comprehensive evaluation of complex interactions among cancer cells, immune cells, and local immunosuppressive elements in the TIME. METHODS:Whole-exome sequencing and RNA sequencing were carried out on 113 lung cancers. We performed single sample gene set enrichment analysis on TIME-related gene sets to develop a new scoring system (TIME score), consisting of T-score (umor proliferation), I-score (antitumor mmunity) and S-score (immunouppression). Lung cancers were classified according to a combination of high or low T-score, I-score, and S-scores (eight groups; G1-8). Clinical and genomic features, and immune landscape were investigated among eight groups. The external data sets of 990 lung cancers from The Cancer Genome Atlas and 76 melanomas treated with immune checkpoint inhibitors (ICI) were utilized to evaluate TIME scoring and explore prognostic and predictive accuracy. RESULTS:The representative histological type including adenocarcinoma and squamous cell carcinoma, and driver mutations such as and mutations were different according to the T-score. The numbers of somatic mutations and predicted neoantigens were higher in T (G5-8) than T (G1-4) tumors. Immune selection pressure against neoantigen expression occurred only in T and was dampened in T/I (G5-6), possibly due to a reduced number of T cells with a high proportion of tumor specific but exhausted cells. T/I/S (G5) displayed the lowest immune responses by additional immune suppressive mechanisms. The T-score, I-score and S-scores were independent prognostic factors, with survival curves well separated into eight groups with G5 displaying the worst overall survival, while the opposite group T/I/S (G4) had the best prognosis. Several oncogenic signaling pathways influenced on T-score and I-scores but not S-score, and PI3K pathway alteration correlated with poor prognosis in accordance with higher T-score and lower I-score. Moreover, the TIME score predicted the efficacy of ICI in patients with melanoma. CONCLUSION:The TIME score capturing complex interactions among tumor proliferation, antitumor immunity and immunosuppression could be useful for prognostic predictions or selection of treatment strategies in patients with lung cancer. 10.1136/jitc-2021-003765
    Prognostic Value and Immunological Characteristics of a Novel RNA Binding Protein Signature in Cutaneous Melanoma. Frontiers in genetics BACKGROUND:The existing studies indicate that RNA binding proteins (RBPs) are closely correlated with the genesis and development of cancers. However, the role of RBPs in cutaneous melanoma remains largely unknown. Therefore, the present study aims to establish a reliable prognostic signature based on RBPs to distinguish cutaneous melanoma patients with different prognoses and investigate the immune infiltration of patients. METHODS:After screening RBPs from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, Cox and least absolute shrinkage and selection operator (LASSO) regression analysis were then used to establish a prediction model. The relationship between the signature and the abundance of immune cell types, the tumor microenvironment (TME), immune-related pathways, and immune checkpoints were also analyzed. RESULTS:In total, 7 RBPs were selected to establish the prognostic signature. Patients categorized as a high-risk group demonstrated worse overall survival (OS) rates compared to those of patients categorized as a low-risk group. The signature was validated in an independent external cohort and indicated a promising prognostic ability. Further analysis indicated that the signature wasan independent prognostic indicator in cutaneous melanoma. A nomogram combining risk score and clinicopathological features was then established to evaluate the 3- and 5-year OS in cutaneous melanoma patients. Analyses of immune infiltrating, the TME, immune checkpoint, and drug susceptibility revealed significant differences between the two groups. GSEA analysis revealed that basal cell carcinoma, notch signaling pathway, melanogenesis pathways were enriched in the high-risk group, resulting in poor OS. CONCLUSION:We established and validated a robust 7-RBP signature that could be a potential biomarker to predict the prognosis and immunotherapy response of cutaneous melanoma patients, which provides new insights into cutaneous melanoma immunotherapeutic strategies. 10.3389/fgene.2021.723796
    Risk stratification of cutaneous melanoma reveals carcinogen metabolism enrichment and immune inhibition in high-risk patients. Li Xia,Cai Yunpeng Aging Cutaneous melanoma (CM) is the most lethal form of skin cancer. Risk assessment should facilitate stratified surveillance and guide treatment selection. Here, based on the mRNA-seq data from 419 CM patients in the Cancer Genome Atlas (TCGA), we developed a prognostic 21-gene signature to distinguish the outcomes of high- and low-risk patients, which was further validated in two external cohorts. The signature achieved a higher C-index as compared with other known biomarkers and clinical characteristics in both the TCGA and validation cohorts. Notably, in high-risk patients the expression levels of three driver genes, , , and in the MAPK pathway, were lower but exhibited a stronger positive correlation as compared with low-risk patients. Moreover, the genes involved in nicotinamide adenine dinucleotide metabolism were negatively correlated with the expression of in the high-risk group. Function analysis revealed that the upregulated genes in the high-risk group were enriched in the cytochrome P450-mediated metabolism of chemical carcinogens. Furthermore, the low-risk group had high levels of gamma delta T cells infiltration, while regulatory T cells were accumulated in the high-risk group. The present study offers a promising new prognostic signature for CM, and provides insight into the mechanisms of melanoma progression. 10.18632/aging.103734
    PRMT5 control of cGAS/STING and NLRC5 pathways defines melanoma response to antitumor immunity. Kim Hyungsoo,Kim Heejung,Feng Yongmei,Li Yan,Tamiya Hironari,Tocci Stefania,Ronai Ze'ev A Science translational medicine Protein arginine methyltransferase 5 (PRMT5) controls diverse cellular processes and is implicated in cancer development and progression. Here, we report an inverse correlation between PRMT5 function and antitumor immunity. expression was associated with an antitumor immune gene signature in human melanoma tissue. Reducing PRMT5 activity antagonized melanoma growth in immunocompetent but not immunocompromised mice. PRMT5 methylation of IFI16 [interferon-γ (IFN-γ)-inducible protein 16] or its murine homolog IFI204, which are components of the cGAS/STING (stimulator of IFN genes) pathway, attenuated cytosolic DNA-induced IFN and chemokine expression in melanoma cells. PRMT5 also inhibited transcription of the gene encoding NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5), a protein that promotes the expression of genes implicated in major histocompatibility complex class I (MHCI) antigen presentation. PRMT5 knockdown augmented IFN and chemokine production and increased MHCI abundance in melanoma. Increased expression of and was associated with decreased melanoma growth in murine models, and increased expression of and correlated with prolonged survival of patients with melanoma. Combination of pharmacological (GSK3326595) or genetic (shRNA) inhibition of PRMT5 with immune checkpoint therapy limited growth of murine melanoma tumors (B16F10 and YUMM1.7) and enhanced therapeutic efficacy, compared with the effect of either treatment alone. Overall, our findings provide a rationale to test PRMT5 inhibitors in immunotherapy-based clinical trials as a means to enhance an antitumor immune response. 10.1126/scitranslmed.aaz5683
    EZH2 Cooperates with DNA Methylation to Downregulate Key Tumor Suppressors and IFN Gene Signatures in Melanoma. Tiffen Jessamy,Gallagher Stuart J,Filipp Fabian,Gunatilake Dilini,Emran Abdullah Al,Cullinane Carleen,Dutton-Register Ken,Aoude Lauren,Hayward Nick,Chatterjee Aniruddha,Rodger Euan J,Eccles Michael R,Hersey Peter The Journal of investigative dermatology The histone methylase EZH2 is frequently dysregulated in melanoma and is associated with DNA methylation and silencing of genes involved in tumor suppression. In this study, we used chromatin immunoprecipitation and sequencing to identify key suppressor genes that are silenced by histone methylation in constitutively active EZH2 mutant melanoma and assessed whether these regions were also sites of DNA methylation. The genes identified were validated by their re-expression after treatment with EZH2 and DNA methyltransferase inhibitors. The expression of putative EZH2 target genes was shown to be highly relevant to the survival of patients with melanoma in clinical datasets. To determine correlates of response to EZH2 inhibitors, we screened a panel of 53 melanoma cell lines for drug sensitivity. We compared RNA sequencing profiles of sensitive to resistant melanoma cells and performed pathway analysis. Sensitivity was associated with strong downregulation of IFN-γ and IFN-α gene signatures that were reversed by treatment with EZH2 inhibitors. This is consistent with EZH2-driven dedifferentiated invasive states associated with treatment resistance and defects in antigen presentation. These results suggest that EZH2 inhibitors may be most effectively targeted to immunologically cold melanoma to both induce direct cytotoxicity and increase immune responses in the context of checkpoint inhibitor immunotherapy. 10.1016/j.jid.2020.02.042
    Direct RIG-I activation in human NK cells induces TRAIL-dependent cytotoxicity toward autologous melanoma cells. Daßler-Plenker Juliane,Paschen Annette,Putschli Bastian,Rattay Stephanie,Schmitz Saskia,Goldeck Marion,Bartok Eva,Hartmann Gunther,Coch Christoph International journal of cancer Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy. 10.1002/ijc.31874
    Genetic Aberrations in the CDK4 Pathway Are Associated with Innate Resistance to PD-1 Blockade in Chinese Patients with Non-Cutaneous Melanoma. Yu Jiayi,Yan Junya,Guo Qian,Chi Zhihong,Tang Bixia,Zheng Bin,Yu Jinyu,Yin Ting,Cheng Zhiyuan,Wu Xiaowen,Yu Huan,Dai Jie,Sheng Xinan,Si Lu,Cui Chuanliang,Bai Xue,Mao Lili,Lian Bin,Wang Xuan,Yan Xieqia,Li Siming,Zhou Li,Flaherty Keith T,Guo Jun,Kong Yan Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:PD-1 checkpoint blockade immunotherapy induces long and durable response in patients with advanced melanoma. However, only a subset of patients with melanoma benefit from this approach. The mechanism triggering the innate resistance of anti-PD-1 therapy remains unclear. Whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) analyses were performed in a training cohort ( = 31) using baseline tumor biopsies of patients with advanced melanoma treated with the anti-PD-1 antibody. Copy-number variations (CNVs) for the genes , and were assayed using a TaqMan copy-number assay in a validation cohort ( = 85). The effect of CDK4/6 inhibitors combined with anti-PD-1 antibody monotherapy was evaluated in PD-1-humanized mouse (C57BL/6-hPD-1) and humanized immune system (HIS) patient-derived xenograft (PDX) models. RESULTS:WES revealed several significant gene copy-number gains in the patients of no clinical benefit cohort, such as 12q14.1 loci, which harbor . The association between gain and innate resistance to anti-PD-1 therapy was validated in 85 patients with melanoma ( < 0.05). RNA-Seq analysis of -normal cell lines and -normal tumors showed altered transcriptional output in TNFα signaling via NF-κB, inflammatory response, and IFNγ response gene set. In addition, CDK4/6 inhibitor (palbociclib) treatment increased PD-L1 protein levels and enhanced efficacy ( < 0.05) in the C57BL/6-hPD-1 melanoma cell and the HIS PDX model. CONCLUSIONS:In summary, we discovered that genetic aberrations in the CDK4 pathway are associated with innate resistance to anti-PD-1 therapy in patients with advanced melanoma. Moreover, our study provides a strong rationale for combining CDK4/6 inhibitors with anti-PD-1 antibody for the treatment of advanced melanomas. 10.1158/1078-0432.CCR-19-0475
    Conserved Interferon-γ Signaling Drives Clinical Response to Immune Checkpoint Blockade Therapy in Melanoma. Grasso Catherine S,Tsoi Jennifer,Onyshchenko Mykola,Abril-Rodriguez Gabriel,Ross-Macdonald Petra,Wind-Rotolo Megan,Champhekar Ameya,Medina Egmidio,Torrejon Davis Y,Shin Daniel Sanghoon,Tran Phuong,Kim Yeon Joo,Puig-Saus Cristina,Campbell Katie,Vega-Crespo Agustin,Quist Michael,Martignier Christophe,Luke Jason J,Wolchok Jedd D,Johnson Douglas B,Chmielowski Bartosz,Hodi F Stephen,Bhatia Shailender,Sharfman William,Urba Walter J,Slingluff Craig L,Diab Adi,Haanen John B A G,Algarra Salvador Martin,Pardoll Drew M,Anagnostou Valsamo,Topalian Suzanne L,Velculescu Victor E,Speiser Daniel E,Kalbasi Anusha,Ribas Antoni Cancer cell We analyze the transcriptome of baseline and on-therapy tumor biopsies from 101 patients with advanced melanoma treated with nivolumab (anti-PD-1) alone or combined with ipilimumab (anti-CTLA-4). We find that T cell infiltration and interferon-γ (IFN-γ) signaling signatures correspond most highly with clinical response to therapy, with a reciprocal decrease in cell-cycle and WNT signaling pathways in responding biopsies. We model the interaction in 58 human cell lines, where IFN-γ in vitro exposure leads to a conserved transcriptome response unless cells have IFN-γ receptor alterations. This conserved IFN-γ transcriptome response in melanoma cells serves to amplify the antitumor immune response. Therefore, the magnitude of the antitumor T cell response and the corresponding downstream IFN-γ signaling are the main drivers of clinical response or resistance to immune checkpoint blockade therapy. 10.1016/j.ccell.2020.08.005
    A whole-blood RNA transcript-based gene signature is associated with the development of CTLA-4 blockade-related diarrhea in patients with advanced melanoma treated with the checkpoint inhibitor tremelimumab. Friedlander Philip,Wood Kevin,Wassmann Karl,Christenfeld Alan M,Bhardwaj Nina,Oh William K Journal for immunotherapy of cancer BACKGROUND:Anti-CTLA-4 immune checkpoint blockade is associated with immune-related adverse events (irAEs). Grade 3-4 diarrhea/colitis is the most frequent irAE requiring treatment discontinuation. Predicting high-risk diarrhea/colitis patients may facilitate early intervention, limit irAE severity, and extend treatment duration. No biomarkers currently predict for anti-CTLA-4 immunotherapy related severe diarrhea. METHODS:Whole-blood was collected pre-treatment and 30 days post-treatment initiation from patients with stage III or IV unresectable melanoma who received 15 mg/kg tremelimumab at 90 day intervals in two clinical trials. The discovery dataset was a phase II study that enrolled 150 patients between December 2005 and November 2006. The validation dataset was a phase III study that enrolled 210 patients between March 2006 and July 2007. RT-PCR was performed for 169 genes associated with inflammation, immunity, CTLA-4 pathway and melanoma. Gene expression was correlated with grade 0-1 versus grade 2-4 diarrhea/colitis development. RESULTS:Pre-treatment blood obtained from the discovery dataset (N = 150) revealed no gene predictive of diarrhea/colitis development (p < 0.05). A 16-gene signature (CARD12, CCL3, CCR3, CXCL1, F5, FAM210B, GADD45A, IL18bp, IL2RA, IL5, IL8, MMP9, PTGS2, SOCS3, TLR9 and UBE2C) was identified from 30 days post-tremelimumab initiation blood that discriminated patients developing grade 0-1 from grade 2-4 diarrhea/colitis. The 16-gene signature demonstrated an AUC of 0.814 (95% CI 0.743 to 0.873, p < 0.0001), sensitivity 42.9%, specificity 99.2%, positive predictive value (PPV) 90.0%, and negative predictive value (NPV) 91.4%. In the validation dataset (N = 210), the 16-gene signature discriminated patients developing grade 0-1 from grade 2-4 diarrhea/colitis with an AUC 0.785 (95% CI 0.723 to 0.838, p < 0.0001), sensitivity 57.1%, specificity 84.4%, PPV 57.1% and NPV 84.4%. CONCLUSION:This study identifies a whole-blood mRNA signature predictive of a clinically relevant irAE in patients treated with immune checkpoint blockade. We hypothesize that immune system modulation induced by immune checkpoint blockade results in peripheral blood gene expression changes that are detectable prior to clinical onset of severe diarrhea. Assessment of peripheral blood gene expression changes in patients receiving anti-PD-1/PD-L1 immunotherapy, or combination anti-CTLA4 and anti-PD-1/PD-L1 immunotherapy, is warranted to provide early on-treatment mechanistic insights and identify clinically relevant predictive biomarkers. TRIAL REGISTRATION:Clinicaltrials.gov , NCT00257205 , registered 22 November 2005. 10.1186/s40425-018-0408-9
    RNA-Containing Immune Complexes Formed by Anti-Melanoma Differentiation Associated Gene 5 Autoantibody Are Potent Inducers of IFN-α. Wang Kaiwen,Zhao Jiangfeng,Wu Wanlong,Xu Wenwen,Sun Shuhui,Chen Zhiwei,Fu Yakai,Guo Li,Du Hui,Ye Shuang Frontiers in immunology Objective:Anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is a distinctive serology hallmark of dermatomyositis (DM). As an autoantigen, MDA5 is a cytoplasmic RNA recognition receptor. The aim of this study was to address the question of whether the RNA-containing immune complex (IC) formed by MDA5 and anti-MDA5 could activate type I interferon (IFN) response. Method:Patients with anti-MDA5 DM ( = 217), anti-MDA5 DM ( = 68), anti-synthase syndrome (ASyS, = 57), systemic lupus erythematosus (SLE, = 245), rheumatoid arthritis (RA, = 89), and systemic sclerosis (SSc, = 30) and healthy donors (HD, = 94) were enrolled in our studies. Anti-MDA5 antibody was detected by line blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Cytokine profiling was determined by multiplex flow cytometry, and IFN-α was further measured by ELISA. Type I IFN-inducible genes were detected by quantitative PCR (qPCR). RNA-IC binding was analyzed by RNA immunoprecipitation. Plasmacytoid dendritic cells (pDCs) derived from healthy donors were cultivated and stimulated with MDA5 ICs with or without RNase and Toll-like receptor 7 (TLR-7) agonist. The interaction between MDA5 ICs and TLR7 was evaluated by immunoprecipitation and confocal microscopy. Results:According to our in-house ELISA, the presence of anti-MDA5 antibody in 76.1% of DM patients, along with 14.3% of SLE patients who had a lower titer yet positive anti-MDA5 antibody, was related to the high level of peripheral IFN-α. ICs formed by MDA5 and anti-MDA5 were potent inducers of IFN-α TLR-7 in an RNA-dependent manner . Conclusion:Our data provided evidence of the mechanistic relevance between the anti-MDA5 antibody and type I IFN pathway. 10.3389/fimmu.2021.743704
    Identification of m7G-associated lncRNA prognostic signature for predicting the immune status in cutaneous melanoma. Aging RNA modifications, including RNA methylation, are widely existed in cutaneous melanoma (CM). Among epigenetic modifications, N7-methylguanosine (m7G) is a kind of modification at 5' cap of RNA which participate in maintaining the stability of mRNA and various cell biological processes. However, there is still no study concerning the relationship between CM and m7G methylation complexes, METTL1 and WDR4. Here, long non-coding RNA (lncRNAs) and gene expression data of CM from the Cancer Genome Atlas (TCGA) database were retrieved to identify differentially expressed m7G-related lncRNAs connected with overall survival of CM. Then, Cox regression analyses was applied to construct a lncRNA risk signature, the prognostic value of identified signature was further evaluated. As a result, 6 m7G-associated lncRNAs that were significantly related to CM prognosis were incorporated into our prognostic signature. The functional analyses indicated that the prognostic model was correlated with patient survival, cancer metastasis, and growth. Meanwhile, its diagnostic accuracy was better than conventional clinicopathological characteristics. The pathway enrichment analysis showed that the risk model was enriched in several immunity-associated pathways. Moreover, the signature model was significantly connected with the immune subtypes, infiltration of immune cells, immune microenvironment, as well as several m6A-related genes and tumor stem cells. Finally, a nomogram based on the calculated risk score was established. Overall, a risk signature based on 6 m7G-associated lncRNAs was generated which presented predictive value for the prognosis of CM patients and can be further used in the development of novel therapeutic strategies for CM. 10.18632/aging.204151
    ZEB1 transcription factor promotes immune escape in melanoma. Journal for immunotherapy of cancer BACKGROUND:The efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration. Oncogenic alterations of tumor cells have been associated to T cell exclusion. Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy. The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies. We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy. METHODS:We evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses. Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8, CD4 T cells number and their activation state (PD-1, Ki67). ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses. Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes. Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated. RESULTS:Combined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8 T cell infiltration, independently of β-catenin pathway activation. ZEB1 ectopic expression in melanoma cells impairs CD8 T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10. Finally, knock-out, by promoting CD8 T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression. CONCLUSIONS:We identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma. TRIAL REGISTRATION NUMBER:NCT02828202. 10.1136/jitc-2021-003484
    Mutations in the IFNγ-JAK-STAT Pathway Causing Resistance to Immune Checkpoint Inhibitors in Melanoma Increase Sensitivity to Oncolytic Virus Treatment. Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:Next-generation sequencing studies and CRISPR-Cas9 screens have established mutations in the IFNγ-JAK-STAT pathway as an immune checkpoint inhibitor (ICI) resistance mechanism in a subset of patients with melanoma. We hypothesized ICI resistance mutations in the IFNγ pathway would simultaneously render melanomas susceptible to oncolytic virus (OV) therapy. EXPERIMENTAL DESIGN:Cytotoxicity experiments were performed with a number of OVs on a matched melanoma cell line pair generated from a baseline biopsy and a progressing lesion with complete loss from a patient that relapsed on anti-PD-1 therapy, in melanoma lines following JAK1/2 RNA interference (RNAi) and pharmacologic inhibition and in knockout (KO) B16-F10 mouse melanomas. Furthermore, we estimated the frequency of genetic alterations in the IFNγ-JAK-STAT pathway in human melanomas. RESULTS:The melanoma line from an anti-PD-1 progressing lesion was 7- and 22-fold more sensitive to the modified OVs, herpes simplex virus 1 (HSV1-dICP0) and vesicular stomatitis virus (VSV-Δ51), respectively, compared with the line from the baseline biopsy. RNAi, JAK1/2 inhibitor studies, and studies of KOs B16-F10 melanomas revealed a significant increase in VSV-Δ51 sensitivity with JAK/STAT pathway inhibition. Our analysis of The Cancer Genome Atlas data estimated that approximately 11% of ICI-naïve cutaneous melanomas have alterations in IFNγ pathway genes that may confer OV susceptibility. CONCLUSIONS:We provide mechanistic support for the use of OVs as a precision medicine strategy for both salvage therapy in ICI-resistant and first-line treatment in melanomas with IFNγ-JAK-STAT pathway mutations. Our study also supports JAK inhibitor-OV combination therapy for treatment-naïve melanomas without IFN signaling defects.. 10.1158/1078-0432.CCR-20-3365
    A One-Armed Phase I Dose Escalation Trial Design: Personalized Vaccination with IKKβ-Matured, RNA-Loaded Dendritic Cells for Metastatic Uveal Melanoma. Koch Elias A T,Schaft Niels,Kummer Mirko,Berking Carola,Schuler Gerold,Hasumi Kenichiro,Dörrie Jan,Schuler-Thurner Beatrice Frontiers in immunology Uveal melanoma (UM) is an orphan disease with a mortality of 80% within one year upon the development of metastatic disease. UM does hardly respond to chemotherapy and kinase inhibitors and is largely resistant to checkpoint inhibition. Hence, further therapy approaches are urgently needed. To improve clinical outcome, we designed a trial employing the 3 generation personalized IKKβ-matured RNA-transfected dendritic cell (DC) vaccine which primes T cells and in addition activates NK cells. This ongoing phase I trial [NCT04335890 (www.clinicaltrials.gov), Eudract: 2018-004390-28 (www.clinicaltrialsregister.eu)] investigates patients with treatment-naive metastatic UM. Monocytes are isolated by leukapheresis, differentiated to immature DCs, matured with a cytokine cocktail, and activated the NF-κB pathway by electroporation with RNA encoding a constitutively active mutant of IKKβ. Three types of antigen-RNA are co-electroporated: i) amplified mRNA of the tumor representing the whole transcriptome, ii) RNA encoding driver mutations identified by exome sequencing, and iii) overexpressed non-mutated tumor antigens detected by transcriptome sequencing. This highly personalized DC vaccine is applied by 9 intravenous infusions in a staggered schedule over one year. Parallel to the vaccination, standard therapy, usually an immune checkpoint blockade (ICB) as mono (anti-PD-1) or combined (anti-CTLA4 and anti-PD-1) regimen is initiated. The coordinated vaccine-induced immune response encompassing tumor-specific T cells and innate NK cells should synergize with ICB, perhaps resulting in measurable clinical responses in this resistant tumor entity. Primary outcome measures of this trial are safety, tolerability and toxicity; secondary outcome measures comprise overall survival and induction of antigen-specific T cells. 10.3389/fimmu.2022.785231
    Integrative analysis of long extracellular RNAs reveals a detection panel of noncoding RNAs for liver cancer. Zhu Yumin,Wang Siqi,Xi Xiaochen,Zhang Minfeng,Liu Xiaofan,Tang Weina,Cai Peng,Xing Shaozhen,Bao Pengfei,Jin Yunfan,Zhao Weihao,Chen Yinghui,Zhao Huanan,Jia Xiaodong,Lu Shanshan,Lu Yinying,Chen Lei,Yin Jianhua,Lu Zhi John Theranostics Long extracellular RNAs (exRNAs) in plasma can be profiled by new sequencing technologies, even with low abundance. However, cancer-related exRNAs and their variations remain understudied. We investigated different variations (i.e. differential expression, alternative splicing, alternative polyadenylation, and differential editing) in diverse long exRNA species (e.g. long noncoding RNAs and circular RNAs) using 79 plasma exosomal RNA-seq (exoRNA-seq) datasets of multiple cancer types. We then integrated 53 exoRNA-seq datasets and 65 self-profiled cell-free RNA-seq (cfRNA-seq) datasets to identify recurrent variations in liver cancer patients. We further combined TCGA tissue RNA-seq datasets and validated biomarker candidates by RT-qPCR in an individual cohort of more than 100 plasma samples. Finally, we used machine learning models to identify a signature of 3 noncoding RNAs for the detection of liver cancer. We found that different types of RNA variations identified from exoRNA-seq data were enriched in pathways related to tumorigenesis and metastasis, immune, and metabolism, suggesting that cancer signals can be detected from long exRNAs. Subsequently, we identified more than 100 recurrent variations in plasma from liver cancer patients by integrating exoRNA-seq and cfRNA-seq datasets. From these datasets, 5 significantly up-regulated long exRNAs were confirmed by TCGA data and validated by RT-qPCR in an independent cohort. When using machine learning models to combine two of these validated circular and structured RNAs () with a miRNA () as a panel to classify liver cancer patients from healthy donors, the average AUROC of the cross-validation was 89.4%. The selected 3-RNA panel successfully detected 79.2% AFP-negative samples and 77.1% early-stage liver cancer samples in the testing and validation sets. Our study revealed that different types of RNA variations related to cancer can be detected in plasma and identified a 3-RNA detection panel for liver cancer, especially for AFP-negative and early-stage patients. 10.7150/thno.48206